Method to treat inflammation with humanized anti-IL-8-antibodies

ABSTRACT

Monoclonal antibodies immunospecific for the neutrophil chemotactic factor, IL-8, have been humanized by reshaping the variable regions to conform more closely to human counterparts. These antibodies are useful in immunoassays to detect IL-8 and as ligands on immunoaffinity columns for purification of human IL-8. In addition, the humanized antibodies have an antiinflammatory effect in patients.

This application is a continuation of Ser. No. 08/437,323, filed May 9,1999, now abandoned, which is a continuation of application Ser. No.08/345,145 filed Nov. 28, 1994 now abandoned which is acontinuation-in-part of U.S. Ser. No. 08/303,841 filed Sep. 8, 1994, nowabandoned, the contents of which are incorporated herein by reference.

TECHNICAL FIELD

The invention relates to monoclonal antibodies which are humanized bymodifying the framework structure that supports the determinant regionsof the variable domains. More specifically, the invention relates tomonoclonal antibodies immunospecific for human IL-8 that have humanizedframework regions so as to be compatible with the human immune systemand to intermediates for the production of such antibodies.

BACKGROUND ART

Human IL-8 is a cytokine which has variously been calledneutrophil-activating protein, neutrophil chemotactic factor (NCF) andT-cell chemotactic factor. IL-8 can be secreted by several types ofcells upon appropriate stimulation. IL-8 is secretedby activatedmonocytes and macrophages as well as by embryonic fibroblasts.

IL-8 is known to induce neutrophil migration and to activate functionsof neutrophils such as degranulation, release of superoxide anion andadhesion to the endothelial cell monolayer. There are a number ofconditions that are known to involve leukocyte infiltration intolesions. These include pulmonary diseases such as pulmonary cysticfibrosis, idiopathic pulmonary fibrosis, adult respiratory distresssyndrome, sarcoidosis and empyema; dermal diseases such as psoriasis,rheumatoid arthritis, Crohn's Disease; and in inflammatory bowel disease(McElvaney, N. G. et al. J Clin Invest (1992) 90:1296-1301; Lynch III,J. P. et al. Am Rev Respir Dis (1992) 145:1433-1439; Donnelly, S. C. etal. Lancet (1993) 341:643-647; Car, B. D. et al. Am J Respir Crit CareMed (1994) 149:655-659; Antony, V. B. et al. J Immunol (1993)151:7216-7223; Takematsu, H. et al. Arch Dermatol (1993) 129:74-80;Brennan, F. M. et al. Eur J Immunol (1990) 20:2141-2144; Izzo, R. S. etal. Scand J Gastroenterol (1993) 28:296-300; Izzo, R. S. et al. Am JGastroenterol (1992) 87:1447-1452).

The amino acid sequence characterizing human IL-8 was described byMatsushima, et al. in PCT application WO89/08665. More recently,Yoshimura, T., et al. in Mol Immunol (1989) 26:87-93 showed thatmonocyte-derived IL-8 was evidently variably processed at the N-terminusand that the IL-8 originally disclosed by Matsushima et al. wasaccompanied by two forms of the factor which had seven or fiveadditional amino acids at the N-terminus. The longest form accounted forabout 8%, the next longest form for about 47%, and the shortest form forabout 45% of the total IL-8 derived from monocytes.

WO89/08665 also reports the production of murine monoclonal antibodiesimmunoreactive with this protein (called neutrophil chemotactic factorin this publication). An additional murine monoclonal antibodyimmunospecific for IL-8 and designated WS-4 was prepared and reported byKo, Y. C. et al. J Immunol Meth (1992) 149:227-235. DNA encoding heavyand light chains of this antibody was recovered from the hybridoma whichproduced it, as described below. Additional murine-derived antibodies tohuman IL-8 were reported by Boylan, A. M. et al. J Clin Invest (1992)89:1257-1267 (A5.12.14); in PCT application WO92/04372 (Anti-Pep1 ANDAnti-Pep3) and by Mulligan, M. S. et al. (J Immunol (1993)150:5585-5595) (DM/C7).

WS-4 has been shown to inhibit the binding of rabbit IL-8 to rabbitneutrophils by Harada, A. et al. International Immunol (1993) 5:681-690.Administration of WS-4 to rabbits also reduced ischemia/reperfusioninjury in the lung (Sekido, N. et al. Nature (1993) 365:654-657);reduced LPS-induced dermatitis (Harada et al. (supra)); and amelioratedLPS or IL-1 induced arthritis (Akahoshi, T. et al. Lymphokine andCytokine Res (1994) 13:113-116). Administration of DM/C7 intratracheallyto rats was protective with respect to inflammatory lung injury(Mulligan, M. S., et al. (supra)). Thus, animal models have demonstratedthat antibodies to IL-8 are effective in treating diseases andconditions which are characterized by inflammation.

It would therefore be useful to have antibodies immunoreactive withhuman IL-8 which would be compatible with the human immune system sothat these antibodies could be used as therapeutic agents. It is knownthat murine antibodies are highly immunogenic in humans, thus limitingtheir value as therapeutic agents. Furthermore, murine antibodies havelow circulating half-lives in humans. It would be ideal to prepareanti-IL-8 antibodies which do not raise antibodies against themselves inhuman patients and which retain their effectiveness against human IL-8.

Other nonhuman antibodies have been "humanized" with varying degrees ofsuccess in the past. In a straightforward and simple but incompleteapproach, chimeric antibodies are prepared, generally using recombinanttechniques, which contain nonhuman variable regions and human constantregions. This eliminates the constant region as an immunogen in humanpatients, but the possibility of an immune response to the foreignvariable region remains (LoBuglio, A. F. et al. Proc Natl Acad Sci USA(1989) 86:4220-4224).

A more sophisticated approach focuses not only on providinghuman-derived constant regions, but modifying the variable regions aswell so as to reshape them as closely as possible to human form. It isknown that the variable regions of both heavy and light chains containthree complementarity-determining regions (CDRs) which vary in responseto the antigens in question and determine binding capability, flanked byfour framework regions (FRs) which are relatively conserved in a givenspecies and which putatively provide a scaffolding for the CDRs. Whennonhuman antibodies are prepared with respect to a particular antigen,the variable regions can be "reshaped" or "humanized" by grafting CDRsderived from nonhuman antibody on the FRs present in the human antibodyto be modified. Application of this approach to various antibodies hasbeen reported by Sato, K., et al. Cancer Res (1993) 53:851-856.Riechmann, L., et al. Nature (1988) 332:323-327; Verhoeyen, M., et al.Science (1988) 239:1534-1536; Kettleborough, C. A., et al. ProteinEngineering (1991) 4:773-3783; Maeda, H., et al. Human AntibodiesHybridoma (1991) 2:124-134; Gorman, S. D., et al. Proc Natl Acad Sci USA(1991) 88:4181-4185; Tempest, P. R., et al. Bio/Technology (1991)9:266-271; Co, M. S., et al., Proc Natl Acad Sci USA (1991)88:2869-2873; Carter, P., et al., Proc Natl Acad Sci USA (1992)89:4285-4289; and Co, M. S. et al. J Immunol (1992) 148:1149-1154.

In general, these techniques involve identifying the regions responsiblefor binding and those responsible for supporting these binding domainsand then grafting these CDRs onto human framework regions which supportthese binding regions. It will be evident that it is impossible simplyto extrapolate this approach from one antibody raised against oneantigen to the next. Antibodies with respect to a particular antigenwill require carefully designed and particularly devised modificationsto obtain the desired result.

DISCLOSURE OF THE INVENTION

The invention provides antibodies immunoreactive with human IL-8 whichare compatible with the human immune system so as to be less immunogenicto humans than the murine monoclonal antibodies presently available. Theinvention also provides intermediates for the preparation of theseantibodies or their immunoreactive fragments. Thus, the inventionprovides proteins which comprise the variable regions of the heavy andlight chains of antibodies which are immunospecific for human IL-8wherein these variable regions have human characteristics. In general,these variable regions contain the complementarity determining regions(CDRs) of the pertinent antibody secreted by nonhuman cells but theframework regions (FRs) which characterize human antibodies.

By thus reshaping murine- or other nonhuman-derived immunoglobulins,their immunogenicity in human patients is minimized. Of course, thesehumanized forms, since they retain their immunospecificity, are usefulin standard assay procedures for IL-8 and in IL-8 purification.

Thus, in one aspect, the invention is directed to a monoclonal antibodyimmunoreactive with human IL-8 wherein the framework regions (FRs) ofthe variable regions of said antibody and the constant regions of saidantibody are compatible with the human immune system. More specifically,the invention is directed to a monoclonal antibody or immunoreactivefragment thereof, immunoreactive with human IL-8 and compatible with thehuman immune system, wherein the framework regions (FRs) of the variableregions of said antibody or fragment and any constant regions of saidantibody or fragment are of human origin. The invention is also directedto intermediates in the preparation of these antibodies and torecombinant materials useful to produce the antibodies and theirintermediates. The invention is also directed to methods to utilizethese antibodies. The antibodies can be used therapeutically,diagnostically, and as reagents for purification of human or other IL-8.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents expression plasmids HEF-VL-gκ and HEF-VH-gγ1comprising a human elongation factor 1α (HEF-1α) promoter/enhancersystem, useful for the expression of light chain and heavy chain of thepresent antibody, respectively.

FIG. 2 is a graph showing a result of ELISA for confirmation of anability to bind to human IL-8 of the chimeric WS-4 antibody (chL/chH)produced in the supernatant of COS cells.

FIGS. 3A-3B show a diagram of the construction of the first versions ofa reshaped human WS-4 heavy chain and light chain variable regions.

FIG. 4 is a graph showing a result of ELISA testing the ability to bindto human IL-8 of the reshaped light chain (RVLa) or heavy chain (RVHa).The antibodies chL/chH, RVLa/chH and chL/RVHa were produced in thesupernatant of COS cells.

FIG. 5 is a graph showing a result of ELISA testing the ability to bindto human IL-8 of the reshaped antibodies comprising the. first version"a" of light chain (RVLa) and various versions of heavy chain-producedin the supernatant of COS cells.

FIG. 6 is a graph showing a result of ELISA testing the ability to bindto human IL-8 of the reshaped antibodies comprising the second version"b" of light chain (RVLb) and various versions of heavy chain producedin the supernatant of COS cells.

FIG. 7 is a graph showing a result of ELISA testing the ability to bindto human IL-8 of the purified chimeric (chL/chH) and reshaped (RVLa/RVHgand RVLb/RVHg) antibodies.

FIG. 8 is a graph showing the results of a receptor binding inhibitionassay. This assay tests the ability of purified control human IgG, achimeric antibody (chL/chH), reshaped antibodies (RVLa/RVHg andRVLb/RVHg) and the original WS-4 antibody to block binding of IL-8 toits receptor.

MODES OF CARRYING OUT THE INVENTION

The invention is directed to antibodies or fragments immunospecific forhuman IL-8 which have sufficient human characteristics so that theirimmunogenicity in human systems is lowered with respect to thecorresponding antibodies derived from other species. Thus, theantibodies or immunoreactive fragments of the invention are compatiblewith the human immune system. By "compatible with the human immunesystem" is meant that the antibodies or fragments of the invention donot elicit a substantial immune response when administered to humans ascompared to unmodified forms of nonhuman antibodies containing the samecomplementarity-determiling regions (CDRs). Eliciting an immune responseis clearly undesirable as antibodies raised against therapeuticallyadministered materials undermine the effectiveness of the administeredmaterials and in addition may provoke unwanted side-effects due tostimulation of the immune system per se. While the antibodies andfragments of the invention may not, of course, be completely neutralwith respect to an immune response in a specific individual, theireffect on the immune system of an individual will be substantially lessthan that elicited by corresponding nonhuman antibodies in theirunmodified forms.

With respect to immunoreactive fragments, it is, of course, well knownthat immunospecificity may be retained using only the F_(ab) or F_(ab')or F.sub.(ab').sbsb.2 regions of immunoglobulins, since it is thevariable regions represented by these fragments which are responsiblefor antigen specificity. The fragments can be obtained directly usingrecombinant or synthetic methods or may be obtained from the antibodiesof the invention by known suitable cleavage techniques. An additionaland particularly useful type of fragment is an F_(v) fragment whereinthe variable regions of the light and heavy chains are covalently linkedthrough a peptide linker. Variable regions of this type are described byBird, R. E. et al. Trends in Biotechnology (1991) 9:132-137 and byHuston, J. S. et al. Proc Natl Acad Sci USA (1988) 85:5879-5883. Theseregions can be linked head-to-tail so that the F_(v) region can berecombinantly produced as a fusion protein.

One object of the invention is to provide a modified form of nonhumanantibodies which retain their immunospecificity with respect to humanIL-8 but nevertheless are compatible with the human immune system byvirtue of humanized reshaping of the nonantigen binding regions. It isrecognized that various components of these antibodies, and variousprecursor forms are useful as intermediates in obtaining this desiredresult. The heavy- and light-chain portions of the antibodies could, intheory, be prepared separately and added together with optionalsubsequent formation of disulfide bonds or ligation with a syntheticlinker. Similarly, individual portions of the heavy and light chains canbe prepared separately and ligated to form complete sequences. Becauseof this possibility, these components are also subjects of theinvention.

However, it is much more practical to prepare the antibodies or theirfragments recombinantly using the nucleotide sequences encoding themplaced in expression vectors and transfected or transformed intorecombinant host cells. Thus, the expression systems for the heavy andlight chains of the antibodies of the invention are also included withinthe scope of the invention, as are expression vectors for nucleotidesequences for portions of these chains. For antibodies or theirfragments which are multimeric with separate chains linked throughdisulfide linkages, expression systems for the heavy and light chains ortheir variable regions may be disposed on a single vector, or separatevectors may be used for heavy and light chains. For construction of thesingle chain F_(v) fragment, of course, a single vector contains theexpression system for this single-chain form. In addition, isolated andpurified or recombinant nucleotide sequences encoding the relevantportions of the desired heavy and light chains are included within theinvention since these portions can be manipulated to obtain a nucleotidesequence encoding the entire chain.

Also included within the scope of the invention are nucleotide sequencesencoding heavy or light chains which are themselves intermediates in thedesign and production of the monoclonal antibodies of the invention.

Thus, the invention includes the following proteins or peptides andpolynucleotides containing nucleotide sequences encoding them: theindividual CDR regions of nonhuman anti-IL-8 heavy and light chainspreferably those of WS-4; variable regions of heavy and light chainscontaining human FRs and nonhuman CDRs immunoreactive with human IL-8;the foregoing variable regions linked together, e.g., through disulfidelinkages; the foregoing variable regions linked to human constantregions and the antibodies formed by the resultant heavy and lightchains; heavy and light chains containing nonhuman variable regionsimmunoreactive with human IL-8 and human constant regions and antibodieswhich are comprised of these heavy and light chains; and variouschimeras of the foregoing, such as a heavy chain comprising a variableregion with nonhuman CDRs and human FRs plus a human constant regioncoupled to a light chain containing a nonhuman variable region and ahuman constant region wherein the resultant antibody is immunospecificfor human IL-8.

By "immunospecific for human IL-8" is meant that the antibody reactswith a human IL-8 to the substantive exclusion of other human proteins.It is understood that a certain amount of low-level cross-reactivity isexpected, but the meaning of "immunospecific" is well understood in theart. Cross-reactivity may occur with respect to IL-8 of other speciessuch as IL-8 obtained from rabbit. Furthermore, although frequently thediscussion in the present application concerns "lantibody"immunospecific for human IL-8, it is understood, based on the discussionabove, that only a fragment of the antibody which contains the variableregions will be similarly effective. Therefore, unless otherwise evidentfrom the context, "antibody" is intended to include immunoreactivefragments of the antibody, including single-chain versions thereof, suchas the F_(v) fragment discussed above.

Design of Chimeric Variable Regions

Examples 1 and 2 below describe in detail the recovery, cloning andsequencing of DNA encoding the variable region of heavy and light chainsof WS-4 antibody. The variable region of the light chain is shown aspositions 1-107 of SEQ ID NO 27 and that of heavy chain is shown aspositions 1-122 of SEQ ID NO 29. As shown, the leader sequences are alsoincluded. The cloned mature variable light-chain region is a 107-aminoacid protein; that for heavy chain is 122-amino acid protein. The aminoacid positions are numbered in the sequence listing. The deduced aminoacid sequences shown can be used to ascertain the location of therelevant CDRs since the basic structure of variable regions isunderstood. Thus, the location of these regions can be ascertained byanalysis of the amino acid sequence as described by Kabat, E. A., et al.in "Sequences of Protein of Immunological Interest" US-DHHS (1991) andthe later publications described in the background art section above.The four FRs largely adopt a β sheet conformation and the CDRs formloops connecting the FRs. The CDRs in some cases form part of the βsheet structure. The FRs form a scaffolding to hold the CDRs in closeproximity to form the antigen-binding site. Example 3 below describes indetail the assignment of the CDR and FR regions in each chain. These arealso presented in Tables 1 and 2 below. The amino acid sequences foreach of these regions is there shown.

If desired, intermediate chimeric antibodies containing human constantregions and murine variable regions can be obtained as described inExample 4 hereinbelow to confirm that the correct variable regions havebeen obtained by showing successful binding of the resulting chimericantibody to human IL-8.

The portions of the variable region which represent the frameworkregions (FRs) are then analyzed and compared to the corresponding FRs inhuman immunoglobulins of various subgroups. The subgroup is chosen whichbears the highest homology to the murine FR region. In the case of thekappa light chain of WS-4, this is subgroup I; in the case of heavychain of WS-4, subgroup III.

In general, as used in the present application, constant regions andframework regions "of human origin" refer to these regions of theimmunoglobulin chains which are obtained by replacing amino acidresidues found in the nonhuman antibodies with the correspondingresidues at these positions found in human antibodies. In the case ofconstant regions, generally, the entire human constant region issubstituted for that of the murine or other nonhuman antibody since thisregion is not germane to the immunospecificity of the antibody. For theframework regions, as described below, the human framework regions whichcorrespond most closely in amino acid sequence to those found in thenonhuman antibody of interest are used as the basis for thesubstitution. However, straightforward one-for-one replacement of suchhuman framework regions for the nonhuman ones is seldom possible; it isnecessary to design specific replacement strategies which result inframework regions more closely resembling those of human origin than dothe framework regions of the nonhuman immunoglobulin. This isillustrated, for example, in Table 2 where in FR3 restoring thephenylalanine at position 67 provided a better result than substitutinga leucine as occurs in the model; similarly in FR2, substitutions atpositions 41 and 47 were not favored with respect to the model humansequence. On the other hand, these positions contain residuescharacteristic of the human subgroup to which the model human sequencebelongs.

In more detail, the variable regions from the WS-4 antibody werecompared to all known human variable regions found in the NationalBiomedical Research Foundation (NBRF) database of protein sequences. Thelight-chain variable region of WS-4 was most similar to that of humanantibody HAU (Watanabe, S. et al. Hoppe Seyler's Z Physiolog Chem (1990)351:1291-1295) with a 69.2% identity; the heavy-chain variable region ofWS-4 was most similar to that of human antibody VDH26 (Buluwela, L. etal. EMBO J (1988) 7:2003-2010) with 71.4% identity. The percentidentities even to these human variable regions are less than those toother mouse variable regions, thus providing evidence that the variableregions of WS-4 "look like" mouse variable regions rather than humanones.

Comparison of the variable regions from WS-4 to consensus sequences forthe subgroups of human variable regions as defined by Kabat, E. A. etal. 1991 (supra), specifically with respect to the FRs, are as follows:for the light chain, human subgroup I shows 64.4% identity with WS-4,while the remaining subgroups II-IV show only 51.3%, 57.3% and 57.5%,respectively. For the heavy-chain region, human subgroup III showed a62.3% homology to WS-4 while the percentages for subgroups 1 and 2 wereonly 46.9% and 40.9%, respectively. These results are consistent withthose obtained above since HAU belongs to human subgroup I and VDH26 tosubgroup III.

An additional human antibody containing FRs very similar to those ofWS-4 light chain is REI. There were only five different amino acidresidues in the FRs of light-chain variable region of CAMPATH-1H ascompared to original human REI (Palm, W. et al., Hoppe-Seyler's Z.Physiol Chem (1975) 356:167-191 and Epp, O. et al., Biochemistry (1975)14:4943-4952); positions 39, 71, 104, 105, and 107 numbered according toKabat et al., 1987; see Table 1. The two changes at positions 39 and 71were changes back to the amino acids that occurred in the FRs of ratCAMPATH-1H light chain variable region (Riechmann, L. et al., Nature(1988) 332:323-327. The three changes in FR4 (positions 104, 105, and107) were based on a J region from another human kappa light chain and,therefore, do not constitute a deviation from human.

Two versions of reshaped human WS-4 light-chain variable region weredesigned. In the first version (version "a"), the-human FRs wereidentical to the REI-based FRs present in reshaped human CAMPATH-1H(Riechmann et al., 1988, supra) and the mouse CDRs were identical to theCDRs in mouse WS-4 light chain variable region. The second version(version "b") was based on version "a" with only one amino acid changeat position 71 in human FR3. Residue 71 is part of the canonicalstructure for CDR1 of the light chain variable region as defined byChothia C. et al., (J Mol Biol (1987) 196:901-917). The amino acid atthis position is predicted to directly influence the structure of theCDR1 loop of the light chain variable region and, therefore, may wellinfluence antigen binding ability. In the mouse WS-4 light chainvariable region, position 71 is phenylalanine. In version "b" ofreshaped human WS-4 light chain variable region, the phenylalanine atposition 71 was changed to a tyrosine as found in mouse WS-4 light chainvariable region. Table 1 shows the amino acid sequences of the mouseWS-4 light chain variable region, the FRs of REI as modified for use inreshaped human CAMPATH-1H antibody, and the two versions of reshapedhuman WS-4 light chain variable region.

                  TABLE 1                                                         ______________________________________                                        Design of Two Different Versions of Reshaped Human                            WS-4 Light Chain Variable Region                                              ______________________________________                                                      1         2                3                                         12345678901234567890123       45678901234                                WS-4 DIQMTQSPASLSASVGETVTITC       RASEIIYSYLA                                REI  DIQMTQSPSSLSASVGDRVTITC                                                  RVLa DIQMTQSPSSLSASVGDRVTITC       RASEIIYSYLA                                RVLb                                                                          ----------                                                                                  FR1                      CDR1                                             4              5                                                         567890123456789     0123456                                              WS-4 WYQQKQGKSPQLLVY     NAKTLAD                                              REI  WYQQKPGKAPKLLIY                                                          RVLa WYQQKPGKAPKLLIY     NAKTLAD                                              RVLb                                                                          ------                                                                                  FR2             CDR2                                                        6         7         8            9                                         78901234567890123456789012345678   901234567                             WS-4 GVSSRFSGSGSGTQFSLRISSLQPEDFGSYYC   QHHFGFPRT                             REI  GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC                                         RVLa GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC   QHHFGFPRT                             RVLb                                                                          ------------------                                                                                 FR3                  CDR3                                      10                                                                           8901234567                                                               WS-4 FGGGTKLELK     pos 1-107 of SEQ ID NO 27                                 REI  FGQGTKVEIK                                                               RVLa FGQGTKVEIK     pos 1-107 of SEQ ID NO 73                                 RVLb                                                                               pos 1-107 of SEQ ID NO 77                                                        FR4                                                                   ______________________________________                                    

Note: The FRs given for REI are those found in the reshaped humanCAMPATH-1H antibody (Riechmann et al., 1988, supra). The five underlinedamino acid residues in the REI FRs are those that differ from the aminoacid sequence of human REI (Palm, W. et al., 1975, supra, Epp, O. etal., 1975, supra).

The FRs in the mouse WS-4 heavy chain variable region were most similarto the FRs in human heavy chain variable regions belonging to subgroupIII. As discussed above, in comparing the mouse WS-4 heavy chainvariable region to known human heavy chain variable regions, FR1, 2 and3 were most similar to the human heavy chain variable region of VDH26(Buluwela, L. et al., EMBO J (1988) 7:2003-2010), a member of subgroupIII of human heavy chain variable regions. Since the sequence of FR4 ofVDH26 is unknown, FR4 of 4B4 (Sanz, I. et al., J Immunol (1989)142:883-887), also a member of subgroup III of human heavy chainvariable regions, is used. For the construction of reshaped human WS-4heavy chain variable region, these human heavy chain variable regionsare used as a base for designing the reshaped human WS-4 heavy chainvariable region.

Eight versions of reshaped human WS-4 heavy chain variable region weredesigned. In all eight versions, the human FRs were based on the FR1, 2and 3 of VDH26 and FR4 of 4B4 and the mouse CDRs were identical to theCDRs in mouse WS-4 heavy chain variable region. Table 2 shows the aminoacid sequences of mouse WS-4 heavy chain variable region, the FRs 1-3 ofVDH26, the FR 4 of 4B4 and the eight versions of reshaped human WS-4heavy chain variable region.

                  TABLE 2                                                         ______________________________________                                        Design of Reshaped Human WS-4 Heavy Chain Variable                            Region                                                                        ______________________________________                                                       1         2         3                                                123456789012345678901234567890 12345                                    WS-4  EVKLVESGGGLIQPGDSLRLSCVTSGFTFS DYYLS                                    VDH26 EVQLLESGGGLVQPGGSLRLSCAASGFTFS - RVHa EVQLLESGGGLVQPGGSLRLSCAASGFTFS           DYYLS                                                                  RVHb                                                                          RVHc                                                                          RVHd                                                                          ----                                                                          RVHe                                                                          ----                                                                          RVHf                                                                          RVHg                                                                          ----                                                                          RVHh                                                                                              FR1               CDR1                                              4          5            6                                                 67890123456789 012ABC3456789012345                                      WS-4  WVRQPPGKALEWVG LIRNKANGYTREYSASVKG                                      VDH26 WVRQAQGKGLELVG                                                          RVHa  WVRQAQGKGLELVG LIRNKANGYTREYSASVKG                                      RVHb                                                                          W---------------------                                                        RVHc                                                                          P---------------------------                                                  RVHd                                                                          P-----W---------------------                                                  RVHe                                                                          PP-----W-- -------------------                                                RVHf                                                                          P--A--W---------------------                                                  RVHg                                                                          P-----W---------------------                                                  RVHh                                                                          W---------------------                                                                   FR2              CDR2                                                        7         8            9        100                                       67890123456789012ABC345678901234 567890ABC12                            WS-4  RFTISRDDSQSILYLQMNTLRGEDSATYYCAR ENYRYDVELAY                            VDH26 RLTISREDSKNTLYLQMSSLKTEDLAVYYCAR                                        RVHa  RLTISREDSKNTLYLQMSSLKTEDLAVYYCAR ENYRYDVELAY                            RVHb                                                                          ----------                                                                    RVHc                                                                          RVHd                                                                          ----------                                                                    RVHe                                                                          ----------                                                                    RVHf                                                                          ----------                                                                    RVHg                                                                          -------------------------------                                               RVHh                                                                          -------------------------------                                                                    FR3                  CDR3                                           110                                                                      34567890123                                                             WS-4  WGQGTLVTVSA    pos 1-122 of SEQ ID NO 29                                4B4   WGQGTLVTVSS                                                             RVHa  WGQGTLVTVSS    pos 1-122 of SEQ ID NO 41                                RVHb                                                                              pos 1-122 of SEQ ID NO 45                                                 RVHc                                                                              pos 1-122 of SEQ ID NO 49                                                 RVHd                                                                              pos 1-122 of SEQ ID NO 51                                                 RVHe                                                                              pos 1-122 of SEQ ID NO 55                                                 RVHf                                                                              pos 1-122 of SEQ ID NO 59                                                 RVHg                                                                              pos 1-122 of SEQ ID NO 63                                                 RVHh                                                                              pos 1-122 of SEQ ID NO 65                                                           FR4                                                                 ______________________________________                                    

As shown above, Tables 1 and 2 indicate the various constructions thatwere made. The following considerations are also germane.

In the framing regions contained in the human heavy chain, we identifiedpositions 1, 27, 28, 29, 30, 48 and 71 (numbered according to Kabat(supra) in Table 2) as having a possible adverse influence on antigenbinding. Residue 1 is a surface residue located close to the CDR loops;residues 27-30 are predicted by Chothia, C. et al. Nature (1989)342:877-883 as part of the canonical structure of CDR1. Chothia alsopredicts residue 71 to be part of the canonical structure of CDR2. Theabove seven positions were identical in both WS-4 and VDH26.

Residue 47 in this chain, located in FR2, is buried in the dimer of thelight and heavy chains and supports the conformation of the bindingsites according to Padlan, E. A. Mol Immunol (1991) 28:489-498. Thisresidue is Leu in VDH26 and versions b, d, e, f, g and h of the reshapedhuman heavy chain contain Trp at this position. While this substitutionappears to replace the human residue with that normally occurring inWS-4, this position is also Trp in the consensus sequences for humansubgroup III and the region of the subgroup including this position isidentical to that of WS-4. Thus, this replacement should not increasethe immunogenicity of the antibody to humans. Residues 41 and 67 inVDH26 (in FR2 and FR3, respectively) are different from those found inWS-4, and also different from those of human subgroup III. Thereplacement at position 41 from Gln to Pro occurs in versions c-g andversions e and f have an additional replacement near residue 41 (seeTable 2). In addition, versions g and h replace Leu at position 67 withPhe. These replacements, generally, restore the residues found in WS-4.

Construction of DNA Encoding Reshaped Human WS-4 Variable Regions, andProduction of Antibodies

The construction of DNA encoding reshaped human WS-4 variable regions isdescribed in Example 5.

The first versions of DNA encoding reshaped human WS-4 light and heavychain variable regions were synthesized, then sequenced to ensure thatthe entire DNA sequences of version "a" of reshaped human WS-4 light andheavy chain variable regions code for the correct amino acid sequence.Sequences of the reshaped human WS-4 antibody light chain variableregion version "a" and heavy chain variable region version "a" are shownas the mature sequences in SEQ ID NO 73 and SEQ ID NO 41, respectively.

The other versions of the reshaped human WS-4 variable regions wereconstructed using slight modifications of published PCR-mutagenesistechniques (Kamman, M. et al., Nucleic Acid Res (1989) 17:5404). Asdescribed in the design of the reshaped human WS-4 variable regions, oneadditional version (version "b") of the reshaped human WS-4 light chainvariable region was constructed and seven additional versions (versions"b", "c", "d", "e", "f", "g" and "h") of the reshaped human WS-4 heavychain variable region were constructed. These additional versionscontain a series of minor changes from the first versions. These minorchanges in the amino acid sequences were achieved using PCR mutagenesisto make minor changes in the DNA sequences. PCR primers were designedthat would introduce the necessary changes into the DNA sequence.Following a series of PCR reactions, each PCR product was cloned andsequenced to ensure that the changes in the DNA sequence had occurred asplanned. The sequence of the reshaped human WS-4 antibody light chainvariable region version "b" is shown as the mature protein region in SEQID NO 77. Sequences of the reshaped human WS-4 antibody heavy chainvariable region versions "b", "c", "d", "e", "f", "g" and "h" are shownas the mature protein regions in SEQ ID NOs 45, 49, 51, 55, 59, 63 and65, respectively.

Once the DNA sequences of the different versions of reshaped human WS-4variable regions were confirmed by sequencing, the reshaped human WS-4variable regions were subcloned into mammalian cell expression vectorsalready containing human C regions. Reshaped human WS-4 light chainvariable regions were joined to DNA sequences coding for human κ Cregion. Reshaped human WS-4 heavy chain variable regions were joined toDNA sequences coding for human γ-1 C region. Next, all combinations ofthe reshaped human light chain versions "a" and "b" with the heavy chainvariable region versions "a" to "h" were tested for binding to humanIL-8, and as a result, a reshaped human antibody comprising the lightchain version "a" or "b" and the heavy chain version "g" exhibited anability to bind to IL-8 at a same level as that of chimeric WS-4 (FIG.7).

Alternatively, rather than ligating the DNA encoding the restructuredvariable regions to DNA encoding the relevant constant region, thevariable regions can be coexpressed per se to obtain immunoreactivefragments. In still another alternative, the various coding sequencesfor the variable region of the heavy chain--i.e., RVH(a-h) of Table 1and the DNA encoding the variable region of the light chain (i.e.,sequences encoding RVL(a-b) of Table 2 can be amplified, isolated andligated to a nucleotide sequence encoding a peptide linker of 12-19amino acid residues. DNA encoding either the heavy-- or light-chainvariable region can constitute the upstream portion. The resultingconstruct encoding a fusion protein representing F_(v) can be insertedinto appropriate expression systems by conventional methods.

For the production of the present chimeric or reshaped human antibodiesto human IL-8, any appropriate expression system, including eucaryoticcells, for example, animal cells, such as established mammalian celllines, fungal cells, and yeast cells, as well as procaryotic cells, forexample, bacterial cells such as E. coli cells, may be used. Preferablythe present chimeric or reshaped human antibodies are expressed inmammalian cells such as COS cells or CHO cells. In such cases, aconventional promoter useful for expression in mammalian cells can beused. For example, a viral expression system including the humancytomegalovirus immediate early (HCMV) promoter is preferably used. Someexamples of the expression vector containing the HCMV promoter areHCMV-VH-HCγ1, HCMV-VL-HCκ and the like derived from pSV2neo(WO92/19759).

Alternatively, other promoters suitable for gene expression of thepresent invention in mammalian cells can be used. These promotersinclude viral promoters such as retrovirus, polyoma virus, adenovirusand simian virus 40 (SV40) or mammalian cell-derived promoters such ashuman elongation factor 1α (HEF-1α) promoter. For example, SV40 orHEF-1α promoters are available according to the methods described byMulligan, R. C. et al. (Nature (1979) 277:108-114) or by Mizushima, S.et al. (Nucleic Acids Res (1990) 18:5322), respectively.

The example for the present invention is HEF-1α promoter. The humanpolypeptide chain elongation factor 1α (HEF-1α) is one of the mostabundant proteins and expressed in most cells. The transcriptionalactivity of the human EF-1α promoter-enhancer is about 100-fold strongerthan that of the SV40 early promoter-enhancer (D. W. Kim et al., Gene(1990) 91:217-223 and T. Uetsuki et al., J Biol Chem (1989)264:5791-5798). The expression vectors containing HEF-1α promoterinclude HEF-VH-gγ1 and HEF-VL-gκ as shown in FIG. 1. As a replicationorigin, DNA sequences derived from polyoma virus, adenovirus, SV40 orbovine papilloma virus (BPV) are available. Additionally, in order toamplify the number of gene copies in the host cell, the aminoglucoside3'-phosphotransferase gene, thymidine kinase (TK) gene, xanthine-guaninephosphoribosyltransferase (Ecogpt) gene and dihydrofolate reductase(dhfr) gene are available as selection markers.

In summary, the present invention first provides a light chain variableregion and an heavy chain variable region of a nonhuman monoclonalantibody to human IL-8, as well as nucleotide sequences encoding them.These are useful for the construction of a human/nonhuman chimericantibody and reshaped human antibody to human IL-8. The variable regionsare derived from, for example, WS-4. This light chain variable regionhas an amino acid sequence shown in SEQ ID NO 27; and the heavy chainvariable region has the amino acid sequence shown in SEQ ID NO 29. Theseamino acid sequences are natively encoded by nucleotide sequences shownin SEQ ID NOs: 26 and 28, respectively.

The present invention also relates to a chimeric antibody to human IL-8,comprising: (1) a light chain comprising a human light chain C regionand a mouse light chain variable region; and (2) a heavy chaincomprising a human heavy chain C region and a mouse heavy chain variableregion. The mouse light chain variable region and the mouse heavy chainvariable region and its encoding DNA are described above. The humanlight chain C region may be any human light chain C region, and forexample, is human Cκ or Cλ. The human heavy chain C region may be anyhuman heavy chain C region, and for example, is human Cγ1, γ2, γ3 or γ4.

For the production of the chimeric antibody, two expression vectors,i.e., one comprising a DNA coding for a mouse light chain variableregion and a human light chain C region under the control of anexpression regulatory region such as an enhancer/promoter system, andanother comprising a DNA coding for a mouse heavy chain variable regionand a human heavy chain C region under the expression regulatory regionsuch as an enhancer/promoter system, are constructed. Next, theexpression vectors are cotransfected into host cells such as mammaliancells, and the transfected cells are cultured in vitro or in vivo toproduce a chimeric antibody. Alternatively, the nucleotide sequencesencoding the heavy and light chains are introduced into a singleexpression vector, and the vector is used to transfect into host cells,which are then cultured in vivo or in vitro to produce a desiredchimeric antibody.

The present invention further provides a reshaped antibody or fragmentsto human IL-8, comprising:

(A) a light chain comprising,

(1) optionally, a human light chain C region, and

(2) a light chain variable region comprising light chain FRs of humanorigin, and light chain CDRs of a nonhuman monoclonal antibody to humanIL-8; and

(B) a heavy chain comprising,

(1) optionally a human heavy chain C region, and

(2) a heavy chain variable region comprising heavy chain FRs of humanorigin, and heavy chain CDRs of a nonhuman monoclonal antibody to humanIL-8.

The reshaped antibody may be in conventional multimeric form or may bein a single-chain version, such as the F_(v) fragment.

In a preferred embodiment, the light chain CDRs have amino acidsequences shown in SEQ ID NO 27 wherein the amino acid sequences of theCDRs are defined in Table 1; the heavy chain CDRs have amino acidsequences shown in SEQ ID NO 29 wherein the amino acid sequences of theCDRs are defined in Table 2; human light chain FRs are derived from theREI; human heavy chain FR1, 2 and 3 are derived from these of VDH26, andhuman heavy chain FR4 is derived from that of 4B4; the human light chainC region is human light chain C κ region; and the human heavy chain Cregion is human heavy chain C γ1 region.

In preferred embodiments, the light chain variable region has an aminoacid sequence shown in Table 1 as RVLa or RVLb; and the heavy chainvariable region has an amino acid sequence shown in Table 2 as RVHa,RVHb, RVHc, RVHd, RVHe, RVHf, RVHg or RVHh; with RVHg most preferable asthe heavy chain variable region.

For the production of the reshaped human antibody, two kinds ofexpression vectors, i.e., one comprising a DNA encoding the reshapedlight chain as defined above, under the control of an expressionregulatory region, such as an enhancer/promoter system, and anothercomprising a DNA encoding the reshaped human heavy chain as definedabove, under the expression control region, such as an enhancer/promotersystem, are constructed. Next, the expression vectors are cotransfectedinto host cells such as mammalian cells, and the transfected cells arecultured in vitro or in vivo to produce a reshaped human antibody.Alternatively, a DNA encoding the reshaped human light chain and a DNAencoding the reshaped heavy chain are introduced into a singleexpression vector, and the vector is used to transfect into host cells,which are then cultured in vivo or in vitro to produce a desiredreshaped human antibody.

The chimeric antibody and the reshaped human antibody thus produced canbe isolated and purified with conventional processes, such as Protein Aaffinity chromatography, ion exchange chromatography, gel filtration andthe like.

Utility

The present mouse light chain variable region, reshaped human lightchain variable region, mouse heavy chain variable region and reshapedhuman heavy chain variable region are intrinsically regions which bindto human IL-8, and are therefore considered to be useful as such, or asfusion proteins with other proteins, for preparing pharmaceuticals ordiagnostic agents.

Moreover, the present light chain variable region CDRs and heavy chainvariable region CDRs are intrinsically regions which bind to human IL-8,and are therefore considered to be useful as such or as fusion proteinswith other proteins, for preparing pharmaceuticals or diagnostic agents.

DNA encoding the mouse light chain variable region of the presentinvention is useful for construction of a DNA encoding a chimeric lightchain or a DNA encoding a reshaped human light chain. Similarly, DNAencoding the heavy chain variable region of the present invention isuseful for construction of a DNA encoding a chimeric heavy chain or aDNA encoding a reshaped human heavy chain. Moreover, DNA encoding lightchain variable region CDRs of the present invention is useful forconstruction of a DNA encoding a reshaped human light chain variableregion and a DNA encoding a reshaped human light chain. Similarly, DNAencoding heavy chain variable region CDRs of the present invention isuseful for construction of a DNA encoding a reshaped human heavy chainvariable region and a DNA encoding a reshaped human heavy chain.Additionally, F.sub.(ab').sbsb.2, F_(ab), F_(v) and single chain F_(v),wherein each F_(v) of light chain and heavy chain are linked, can beproduced in a suitable host cell, and then can be used for preparingpharmaceuticals or diagnostic agents.

The antibodies of the present invention are useful not only astherapeutic agents, but also as tools for purification of IL-8 and asreagents for immunoassays. For use as purification tools, the antibodiesare employed in conventional procedures. In general, the antibody or animmunospecific fragment thereof will be used as an affinity ligandcoupled to a solid support. A sample believed to contain IL-8 is placedin contact with the solid support. The support may be a chromatographiccolumn, a derivatized plastic surface, or any adsorbent solid material.The IL-8 is allowed to bind to the immunospecific antibody or fragmentcoupled to solid support and unbound materials are removed. The IL-8 isthen recovered from the solid support using standard procedures.

For use in immunoassays, the antibodies of the invention may be used ina variety of formats including direct or competitive assays for IL-8with a variety of detection systems using well-known protocols generallyknown as RIAs, ELISAs, and the like. The antibodies can also be used aspositive controls in assays for anti-IL-8 antibodies.

For example, for ELISA, appropriate plates are coated with antihumanIL-8 antibody, such as goat antihuman IL-8 polyclonal antibody. Afterblocking the plates, human IL-8 is added to wells. Following washing,purified sample or supernatant from antibody-producing cells containingthe chimeric or reshaped antibody of the present invention is added.After incubation and washing, antihuman Ig antibody coupled to, forexample, alkaline phosphatase, is added. After incubation and washing,enzyme substrate such as p-nitrophenyl phosphate is added and then afteradditional incubation the optical density at 405 nm is measured.

For use in therapy, e.g., to curtail neutrophil activation, afterpurification, evaluation and sterilization of the antibody, the chimericor reshaped antibodies and fragments thereof may be administered byparenteral injection, for example, via intravenous, intramuscular,intraperitoneal, or subcutaneous injection, in a dosage range for humansof 1 to 1000 mg depending on the condition and age of the patient. Thisamounts to roughly 10 μg/kg-20 mg/kg of body weight for animals ingeneral. Of course, optimization of dosage and of administrationprotocols for a particular species and for a particular subject areroutine.

The antibody of the present invention and fragments thereof may beformulated in a conventional manner as described in Remington'sPharmaceutical Science, latest edition, Mack Publishing Company, Easton,Pa. For example, a preparation for injection may be obtained by thedissolving purified antibody in a solvent such as physiological salineor a buffer solution and then adding a substance such as Tween 80,gelatin or human serum albumin (HSA), to the solution to preventnonspecific adsorption by the surfaces of the vessel containing thepreparation. The preparation may be freeze-dried and reconstituted priorto use. Mannitol or glucose may be used as a filler for freeze-drying.

The present invention will be further illustrated by, but is by no meanslimited to, the following Examples.

PREARATION A Construction of Hybridoma WS-4

Starting hybridomas used in the present invention were constructed asfollows. BALB/c mice were immunized with the recombinant human IL-8produced in E. coli. Spleen cells from the mice were fused withP3X63-Ag8.653 mouse myeloma cells using a standard polyethylene glycolmethod. Monoclonal antibodies were screened for specific binding tohuman IL-8 by ELISA using 96-well microtiter plates coated with rhIL-8(Ko, Y. C. et al., J Immunol Methods (1992) 149:227-235).

EXAMPLE 1 Cloning of DNA Encoding Variable Regions of Mouse MonoclonalAntibody to Human IL-8

1. Preparation of Total RNA

Total RNA from hybridoma WS-4 was prepared according to a standardguanidinium thiocyanate/cesium chloride method described by Chirgwin, J.M. et al., Biochemistry (1979) 18:5294-5299.

1×10⁷ cells of the hybridoma WS-4 were completely homogenized in 25 mlof 4 M guanidine thiocyanate (Fluka) solution. The homogenate waslayered over a 5.7 M cesium chloride solution layer in a centrifugetube, which was then centrifuged in a Beckman SW40 rotor at 31000 rpm at20° C. for 14 hours to precipitate the RNA. The RNA precipitate waswashed with 80% ethanol and dissolved in 200 μl of 20 mM Tris-HCl (pH7.5) containing 10 mM EDTA and 0.5% sodium N-lauroylsarcosinate, andafter adding Protenase (Boehringer) thereon to 0.5 mg/ml, incubated at37° C. for 30 minutes in a water bath. The mixture was extracted withphenol and chloroform, and the RNA was precipitated with ethanol. Next,the RNA precipitate was dissolved in 200 μl of 10 mM Tris-HCl (pH 7.5)containing 1 mM EDTA.

2. Extraction of Messenger RNA (mRNA)

Next, poly A⁺ mRNA coding for mouse monoclonal antibody WS-4 heavy chainwas purified from the total RNA using the FAST TRACK mRNA ISOLATION KITversion 3.2 (Invitrogen, USA) according to the manufacturer'sinstructions.

3. Synthesis of Single Stranded cDNA

Single stranded cDNA coding for mouse heavy chain variable region wassynthesized from about 40 ng of the mRNA using the cDNA Cycle Kit(Invitrogen, USA) according to the manufacturer's instructions, thenused for amplification of the cDNA in the same manner. Thereby singlestranded cDNA coding for mouse light chain variable region wassynthesized from about 10 μg of total RNA.

4. Amplification of cDNA Coding for Antibody variable Region by PCRMethod

(1) Amplification of cDNA coding for mouse heavy chain variable region

The primers used for the PCR method were MHV (Mouse Heavy Variable)primers 1 to 12 represented in SEQ ID NOs: 13 to 24 and an MHC (MouseHeavy Constant) primer represented in SEQ ID NO 25 (Jones, S. T. et al.,Bio/Technology (1991) 9:88-89)

First, 100 μl of a PCR solution comprising 10 mM Tris-HCl (pH 8.3), 50mM KCl, 0.1 mM dNTPs (dATP, dGTP, dCTP and dTTP), 1.5 mM MgCl₂, 0.001%(W/V) gelatin, 5 units of DNA polymerase Ampli Taq (Perkin Elmer Cetus),0.25 μM of one of the MHV primers, 1.75 μM MHC primer and 1.5 μl of thereaction mixture of the single-stranded cDNA synthesis was prepared foreach MHV primer, separately. Each PCR reaction mixture was covered with50 μl of mineral oil, then heated at an initial temperature of 94° C.for 3 minutes, and then at 94° C. for 1 minute, 55° C. for 1 minute and72° C. for 1 minute, in this order. After this temperature cycle wasrepeated 30 times, the reaction mixture was further incubated at 72° C.for 10 minutes.

(2) Amplification of cDNA coding for mouse light chain variable region

As primers for the PCR, MKV (Mouse Kappa Variable) primers 1 to 11represented in SEQ ID NOs: 1 to 11 and an MKC (Mouse Kappa Constant)primer represented in SEQ ID NO 12 (Jones, S. T. et al., Bio/Technology(1991) 9:88-89) were used.

Amplification was carried out according to the same procedure asdescribed for the amplification of the heavy chain variable region genein section 4 (1) except that 0.25 μM of each MKV primer was mixed with 3μM MKC primer and 2 μl of the reaction mixture of the single-strandedcDNA synthesis were used.

5. Purification and Digestion of PCR Products

The DNA fragments amplified by the PCR as described above were purifiedby agarose gel electrophoresis using a 1.5% low melting temperatureagarose gel (Sigma). Each agarose piece containing DNA fragments ofabout 450 bp of heavy chain variable region or about 400 bp of lightchain variable region in length was excised and melted at 65° C. for 5minutes, and an equal volume of 20 mM Tris-HCl (pH 7.5) containing 2 mMEDTA and 200 mM NaCl was added thereon. The mixture was extracted withphenol and chloroform, and the DNA was recovered by ethanolprecipitation. Next, the DNA precipitate was digested with 5 units ofXmaI (New England Biolabs), in the buffer supplied with the enzyme (10mM MgCl₂, 1 mM dithiothreitol, 10 mM Tris-HCl (Ph 7.9)) at 37° C. for 3hours, and subsequently digested with 40 units of SalI (Takara Shuzou)in the buffer supplied with the enzyme, at 37° C. for 2 hours. Thedigestion mixture was extracted with phenol and chloroform, and the DNAwas recovered by ethanol precipitation. The resulting DNA fragments wereseparated by agarose gel electrophoresis using low melting agarose(Sigma). Each agarose piece containing DNA fragments was excised andmelted at 65° C. for 5 minutes, and an equal volume of 20 mM Tris-HCl(pH 7.5) containing 2 mM EDTA and 200 m NaCl was added thereon. Themixture was extracted with phenol and chloroform, and the DNA fragmentwas recovered by ethanol precipitation and dissolved in 10 mM Tris-HCl(pH 7.5) containing 1 mM EDTA.

In this manner, a DNA fragment comprising a gene coding for a mouse κlight chain variable region, and a DNA fragment comprising a gene codingfor a mouse heavy chain variable region were obtained. Both of the aboveDNA fragments had a SalI cohesive end at the 5'-end thereof and an XmaIcohesive end at the 3'-end thereof.

6. Ligation and Transformation

The SalI-XmaI DNA fragment comprising a gene coding for a mouse κ lightchain variable region, prepared as described above, was ligated with apUC19 vector (Takara Shuzou). The pUC19 vector was prepared by digestingplasmid pUC19 with XmaI, SalI, and diphosphorylating with E. coli C75alkaline phosphatase (Takara Shuzou) in the buffer supplied with theenzyme. After extraction with phenol and chloroform, about 0.3 μg of theSalI-XmaI DNA fragment was ligated with about 0.1 μg of a pUC19 vectorwith 1 units of T4 DNA ligase (GIBCO BRL), at 16° C. for 4 hours.

Next, 5 μl of the above ligation mixture was added to 50 μl of competentcells of E. coli DH5α (GIBCO BRL), and the cells were incubated for 30minutes on ice, for 1 minute at 42° C., and again for 1 minute on ice.After adding 400 μl of 2×YT medium (Molecular Cloning: A LaboratoryManual, Sambrook et al., Cold Spring Habor Laboratory Press, 1989), thecell suspension was incubated at 37° C. for one hour, and inoculatedonto an 2×YT agar plate (Molecular Cloning: A Laboratory Manual,Sambrook et al., Cold Spring Habor Laboratory Press, 1989) containing 50μg/ml ampicillin (Meiji confectionery), which was then incubated at 37°C. overnight to obtain E. coli transformants. Prior to the incubation,the surface of 2×YT agar plate was covered with X-Gal(5-bromo-4-chloro-3-indolyl-β-D-galactoside, Takara Shuzou) as aselection marker.

The transformant was cultured in 10 ml of 2×YT medium containing 50μg/ml ampicillin, at 37° C. overnight, and the plasmid DNA was preparedfrom the culture with a QIAGEN plasmid mini kit (QIAGEN) according tothe manufacturer's instruction. The thus-obtained plasmid containing agene coding for a mouse κ light chain variable region derived from thehybridoma WS-4, was designated pUC-WS4-VL.

According to the same procedure as described above, except for usingcompetent cells of E. coli JM109 (Nippon Gene), a plasmid containing agene coding for a mouse heavy chain variable region derived from thehybridoma WS-4 was constructed from the SalI-XmaI DNA fragment, anddesignated pUC-WS4-VH.

EXAMPLE 2 Sequencing of DNA

Nucleotide sequences of a cDNA coding region in the above-mentionedplasmids were determined using an Automatic DNA sequencer 373A and TaqDye Deoxy Terminator Cycle Sequence kit (Applied Biosystems) accordingto the manufacturer's instructions. The nucleotide sequences of the cDNAcoding region in pUC-WS4-VL and pUC-WS4-VH are shown in SEQ ID NOs 26and 28, respectively.

EXAMPLE 3 Determination of CDRs

The general structures of the light chain and heavy chain variableregions are similar to each other, wherein the 4 framework regions (FRs)are linked through 3 hypervariable regions, i.e., complementaritydetermining regions (CDRs). While amino acid sequences in the FRs arerelatively well-conserved, amino acid sequences in CDRs are highlyvariable (Kabat, E. A. et al., "Sequences of Proteins of ImmunologicalInterest", U.S. Dept. Health and Human Services 1991).

On the basis of the above-determined amino acid sequences of variableregions of mouse monoclonal antibodies to human IL-8, and according toKabat, E. A. et al., "Sequences of Proteins of Immunological Interest",U.S. Dept. Health and Human Services 1991, CDRs of each variable regionof mouse monoclonal antibodies to human IL-8 were determined as shown inTable 3.

                  TABLE 3                                                         ______________________________________                                        Plasmid   SEQ ID NO  CDR1     CDR2   CDR3                                     ______________________________________                                        pUC-WS4-VL                                                                              27         24-34    50-56  89-97                                    pUC-WS4-VH                                                                              29         31-35    50-68  101-111                                  ______________________________________                                    

EXAMPLE 4 Confirmation of Expression of Cloned cDNA (Construction ofChimeric WS-4 Antibody)

1. Construction of Expression Plasmid

In order to construct expression vectors for chimeric WS-4 antibody, thecDNA clones pUC-WS4-VH and pUC-WS4-VL, coding for the mouse WS4 heavychain variable region and light chain variable region, respectively,were modified by a PCR technique, and then introduced into the HEFexpression vectors (referred to WO92/19759 and FIG. 1). Two sets ofprimers were designed and synthesized. A light chain variable regionbackward primer (SEQ ID NO 30) and heavy chain variable region backwardprimer (SEQ ID NO 31) were designed so that the primers hybridize with aDNA coding for the beginning of the leader sequence, maintain a DNAsequence essential for efficient translation (Kozak, M. et al. J MolBiol (1987) 196:947-950) and form a HindIII cleavage site for cloninginto the expression vector. A light chain variable region forward primer(SEQ ID NO 32) and a heavy chain variable region forward primer (SEQ IDNO 33) were designed so that the primers hybridize with a DNA coding forthe terminal portion of the J region, maintain a DNA sequence essentialfor splicing into the C region and form a BamHI site for joining to thehuman C region in the expression vector.

Each 100 μl PCR mixture contained 20 mM Tris-HCl (pH 8.2), 10 mM KCl, 6mM (NH₄)₂ SO₄, 1% Triton X-100, 1.5 mM MgCl₂, 100 μM dNTPs, 2.5 U of PfuDNA polymerase (STRATAGENE), 100 ng of a template DNA (pUC-WS4-VH orpUC-WS4-VL), 100 pmoles of each primer. Each PCR reaction mixture wascovered with 50 μl of mineral oil, then heated at an initial temperatureof 94° C. for 3 minutes, and then at 94° C. for 1 minute, 55° C. for 1minute and 72° C. for 1 minute, in this order. After this temperaturecycle was repeated 30 times, the reaction mixture was further incubatedat 72° C. for 10 minutes.

Following PCR amplification, the PCR products were purified using 1.5%low melting agarose gel, digested with HindIII and BamHI, and introducedinto the HEF expression vectors containing the human κ or γ1 chain Cregion DNA, and sequenced to confirm that errors were not introducedduring the PCR amplification. The resulting expression vectors weredesignated as HEF-chWS4H-gγ1 and HEF-chWS4L-gκ.

2. Transient Expression in COS Cells

To observe transient expression of a chimeric WS-4 antibody, theexpression vectors constructed as described above were tested in the COScells. The vector DNAS, HEF-chWS4H-gγ1 and HEF-chWS4L-gκ, werecotransfected into COS cells by electroporation using a Gene Pulserapparatus (Bio Rad). Namely, COS cells were suspended inphosphate-buffered saline (PBS) to a cell concentration of 1×10⁷cells/ml, and 10 μg (per each plasmid) of DNA was added to an 0.8 mlaliquot of the suspension. Pulses were applied at 1,500 V and 25 μFcapacitance. After a recovery period of 10 minutes at room temperature,the electroporated cells were added to 15 ml of DMEM (GIBCO BRL)containing 5% γ-globulin-free fetal bovine serum (GIBCO BRL). Afterincubation for 96 hours, a culture supernatant was collected,centrifuged to eliminate cell debris, filtered with a 0.45 μm pore sizedisk filter (Gelman Science) and aseptically stored short-term at 4° C.or long-term at -20° C.

3. ELISA for Human IgG

The supernatant was first determined, by ELISA, if human-like antibodywas produced by the transfected COS cells. By using known amounts ofpurified human IgG as a standard in this assay, it was also possible toestimate an amount of human-like antibody (in this case, chimeric WS-4antibody chL/chH) present in the supernatant from the COS cells.

For this assay, 96-well multiplates (Nunc) were coated with 100 μl of 1μg/ml goat antihuman IgG gamma chain antibody (2 μg/ml in 0.1 M sodiumbicarbonate and 0.02% sodium azide) (TAGO). After blocking with dilutionbuffer (50 mM Tri-HCl (pH 7.2), 1% BSA, 1 mM MgCl₂, 0.15 M NaCl, 0.05%Tween 20 and 0.02% sodium azide), 100 μl of serially diluted supernatantwas added to each well. After incubation and washing, 100 μl of4000-fold diluted alkaline phosphatase-conjugated goat antihuman IgGgamma chain antibody (TAGO Inc, USA) was added to each well. Afterincubation and washing, 100 μl of 1 mg/ml p-nitrophenyl phosphatesubstrate solution (Sigma) was added. After incubation, the opticaldensity at 405 nm was measured. Purified human IgG (Paesel+Lorei) wasused as a standard.

The supernatant from the COS cells transfected with the vectors carryingthe chimeric WS-4 genes was positive for the expression of a human-likeantibody.

4. ELISA Assay for IL-8

Next, the same serially diluted supernatant from the COS cellstransfected with the vectors carrying the chimeric WS-4 genes forchL/chH was assayed-by ELISA for an ability to bind to human IL-8 todetermine whether the produced antibody can bind to the antigen. Forantigen-binding assay, 96-well multiplates (Nunc) were coated with 100μl of goat antihuman IL-8 polyclonal antibody (R & D systems). Followingblocking, 100 μl of recombinant human IL-8 (5 ng/ml, Amersham) was addedto each well. After washing, 100 μl of the supernatant was added. Afterincubation and washing, 100 μl of 4000-fold diluted alkalinephosphatase-conjugated goat antihuman IgG gamma chain antibody (TAGOInc, USA) was added to each well. After incubation and washing, 100 μlof 1 mg/ml p-nitrophenyl phosphate substrate solution (Sigma) was added.After incubation, the optical density at 405 nm was measured. There wasno standard available for this assay.

A result is shown in FIG. 2. The culture supernatant samples exhibited astrong binding to IL-8, and the optical density at 405 nm was changed ina concentration-dependent manner, i.e., dependent on the monoclonalantibody concentration, as shown in FIG. 2, revealing the presence of anantibody to IL-8 in the sample, and suggesting that this chimeric WS-4antibody has the correct structure of the mouse WS-4 monoclonal antibodyvariable region.

EXAMPLE 5 Construction of Reshaped Human WS-4 Antibody Heavy ChainVariable Region

The DNA coding for the first version of the reshaped human WS-4 heavychain variable region was designed as follows. The DNA sequences forFR1, FR2 and FR3 of the VDH26 heavy chain variable region and FR4 of the4B4 heavy chain variable region were linked to the DNA sequences for theCDRs of the mouse WS-4 heavy chain variable region. The DNA sequenceswhich were similar to the consensus sequence for the splice donor sitewere mutated to minimize the possibility of aberrant splicing, and notto change the amino acid sequence. A HindIII restriction site and Kozakconsensus sequence were added to the 5' end and a BamHI restriction siteand a splice donor sequence were added to the 3' end, so that the DNAfragment could be inserted into HEF expression vector. Then the DNAsequence coding for the reshaped human WS-4 heavy chain variable regionwas divided into four oligonucleotides.

The sequences of these oligonucleotides are shown in SEQ ID NOs; 34-37.These oligonucleotides are 113-143 bases in length and have overlappingregions with 20 bases in adjacent oligonucleotides. Among theseoligonucleotides, HF1 (SEQ ID NO 34) and HF3 (SEQ ID NO 35) have senseDNA sequence and HF2 (SEQ ID NO 36) and HF4 (SEQ ID NO 37) haveantisense DNA sequence. These oligonucleotides were synthesized with theautomatic DNA synthesizer (381A, Applied Biosystems). The method forassembly of these oligonucleotides is described below. A process forconstruction of DNA coding for the first versions of reshaped human WS-4antibody light chain variable region is shown in FIG. 3.

Namely, in the first PCR step, 98 μl PCR mixture contained about 100 ngeach of HF1 and HF2, 2.5 U of Pfu DNA polymerase and the buffer suppliedwith the enzyme in a single tube. The other PCR mixture contained 2pmoles each of HF3 and HF4 in another single tube. Each PCR tube wascycled, after denaturation at 94° C. for 3 min, at 94° C. for 2 min, 55°C. for 2 min and 72° C. for 2 min over 2 cycles, and then incubated at72° C. for 10 min, thereby the two oligonucleotides were annealed andextended. The half volume from each reaction was mixed, and incubatedunder the same condition as described above, thereby the two extendedoligonucleotides were annealed and further extended to obtain an entireoligonucleotide. In the second PCR step, 100 pmoles each of the externalprimers, RVH 5' primer (SEQ ID NO 38) and RVH3' primer (SEQ ID NO 39)were added to the first step PCR mixture. The PCR tube was overlaid with50 μl of mineral oil and then cycled, after denaturation at 94° C. for 3min, at 94° C. for 1 min, 55° C. for 1 min and 72° C. for 1 min over 45cycles. The completion of the last cycle was followed by a finalextension at 72° C. for 10 min.

Next, an approximately 450 bp DNA fragment was purified with 1.5% lowmelting agarose, digested with HindIII and BamHI, and then subclonedinto the HEF-VH-gγ1 expression vector (FIG. 1). After DNA sequencing, aplasmid DNA encoding the correctly designed amino acid sequence wasdesignated HEF-RVHa-gγ1. Both the DNA and deduced amino acid sequencescoding for the heavy chain variable region in the expression vectorHEF-RVHa-gγ1 are shown in SEQ ID NO 40.

The other versions of reshaped human WS-4 heavy chain variable region,"b", "c", "d", "e", "f", "g" and "h" were constructed by the followingmethod.

The second version "b" (RVHb) wash constructed using a PCR-basedmutagenesis. The mutagenic primers, LTW-1 (SEQ ID NO 42) and LTW-2 (SEQID NO 43), designed to change the nucleotide T to G at position 197,resulted in the amino acid change of Leu to Trp at position 47(determined by Kabat, E. A) in the FR2. The plasmid HEF-RVHa-gγ1 wasused as the template DNA. The final PCR product was purified, digestedwith HindIII and BamHI, and then cloned into the HEF expression vectorto yield the plasmid, HEF-RVHb-gγ1. Both the DNA and deduced amino acidsequences coding for the heavy chain variable region in the expressionvector HEF-RVHb-gγ1 are shown in SEQ ID NO 44.

The third version "c" (RVHc) and the fourth version "d" (RVHd) were alsoconstructed using a PCR-based mutagenesis. The mutagenic primers, QTP-1(SEQ ID NO 46) and QTP-2 (SEQ ID NO 47), were designed to change thenucleotide A to C at position 179, resulted in the amino acid change ofGln to Pro at position 41 in the FR2. The plasmids HEF-RVHa-gγ1 andHEF-RVHb-gγ1 were used as the template DNA for version "c" and "d",respectively. The final PCR products were purified, digested withHindIII and BamHI, and then cloned into the HEF expression vector toyield the plasmids, HEF-RVHc-gγ1 and HEF-RVHd-gγ1. Both the DNA anddeduced amino acid sequences coding for heavy chain variable region inthe expression vector HEF-RVHc-gγ1 and HEF-RVHd-gγ1 are shown in SEQ IDNO 48 and 50, respectively.

The version "e" (RVHe) was constructed using the mutagenic primers,ATP-1 (SEQ ID NO 52) and ATP-2 (SEQ ID NO 53), designed to change thenucleotide G to C at position 175, resulting in the amino acid change ofAla to Pro at position 40 in the FR2. The plasmids HEF-RVHd-gγ1 was usedas the template DNA for version "e". Thus the HEF expression vectorHEF-RVHe-gγ1 was constructed. Both the DNA and deduced amino acidsequences coding for heavy chain variable region in the expressionvector HEF-RVHe-gγ1 are shown in SEQ ID NO 54.

The version "f" (RVHf) was constructed using the mutagenic primers,GTA-1 (SEQ ID NO 56) and GTA-2 (SEQ ID NO 57) designed to change thenucleotide G to C at position 188, resulting in the amino acid change ofGly to Ala at position 44 in the FR2. The plasmids HEF-RVHd-gγ1 was usedas the template DNA for version "f". Thus the HEF expression vectorHEF-RVHf-gγ1 was constructed. Both the DNA and deduced amino acidsequences coding for the heavy chain variable region in the expressionvector HEF-RVHf-gγ1 are shown in SEQ ID NO 58.

The version "g" and "h" (RVHg and RVHf, respectively) were constructedusing the mutagenic primers, LTF-1 (SEQ ID NO 60) and LTF-2 (SEQ ID NO61), designed to change the nucleotide C to T at position 265, resultingin the amino acid change of Leu to Phe at position 70 in the FR3. Theplasmids HEF-RVHd-gγ1 and HEF-RVHb-gγ1 were used as the templates DNAfor version "g" and "h", respectively. Thus the HEF expression vectorsHEF-RVHg-gγ1 and HEF-RVHh-gγ1 were constructed. Both the DNA and deducedamino acid sequences coding for heavy chain variable region in theexpression vector HEF-RVHg-gγ1 and HEF-RVHh-gγ1 are shown in SEQ ID NO62 and 63, respectively.

Construction of Reshaped Human WS-4 light Chain Variable Region

For the design of the reshaped human WS-4 VL region, the DNA sequencesfor the FRs present in the REI-based reshaped human PM-1 VL region (Satoet al., 1993) were joined with the DNA sequences for the CDRs of themouse WS4 VL region. Then a HindIII restriction site and a Kozakconsensus sequence were added to the 5' end and a BamHI restriction siteand a splice donor sequence were added to the 3' end of the above DNAsequence. Then the DNA sequence coding for the reshaped human WS-4 lightchain variable region was divided into four oligonucleotides.

The sequences of these oligonucleotides are shown in SEQ ID NOs 66-69.The DNA sequence was divided into four oligonucleotides which were106-124 bases in length and had an overlapping regions of 19 or 23bases. Among these four oligonucleotides, LF1 (SEQ ID NO 66) and LF3(SEQ ID NO 68) have sense DNA sequence and LF2 (SEQ ID NO 67) and LF4(SEQ ID NO 69) have antisense DNA sequence. These oligonucleotides weresynthesized and assembled by the same PCR method described in theconstruction of the reshaped human WS-4 heavy chain variable region,except that RVL5' primer (SEQ ID NO 70) and RVL3' primer (SEQ ID NO 71)were used as external primers for amplification.

After the PCR reaction, a DNA fragment of approximately 400 nucleotideswas purified using a 1.5% low melting agarose gel, digested with HindIIIand BamHI, and subcloned into an identically digested pUC vector. AfterDNA sequencing, the HindIII-BamHI fragment encoding the correctlydesigned amino acid sequence was excised from the pUC plasmid DNA andintroduced into the HEF expression vector, yielding HEF-RVLa-gκ. Boththe DNA and deduced amino acid sequences coding for the light chainvariable region in-the expression vector HEF-RVLa-gκ are shown in SEQ IDNO 72.

The second version "b" (RVLb) was constructed using the mutagenicprimers, FTY-1 (SEQ ID NO 73) and FTY-2 (SEQ ID NO 74), designed tochange the nucleotide T to A at position 269, resulting in the aminoacid change of Phe to Tyr at position 71 in the FR3. The plasmidsHEF-RVLa-gκ was used as the template DNA for version "b". Thus the HEFexpression vector HEF-RVLb-gκ was constructed. Both the DNA and deducedamino acid sequences coding for the light chain variable region in theexpression vector HEF-RVLb-gκ are shown in SEQ ID NO 76.

EXAMPLE 6 Expression and Analysis of Different Versions of ReshapedHuman WS-4 Antibody

For evaluation of the first versions of the reshaped human WS-4 antibodylight chain (RVLa) and heavy chain (RVHa) constructed according to themethod described above, each HEF-1α vector expressing reshaped. humanWS-4 light or heavy chains was cotransfected into COS cells with theHEF-1α vectors expressing chimeric WS-4 heavy or light chains,respectively. As a standard control, HEF-1α vectors expressing chimericWS-4 light and heavy chains were also cotransfected into COS cells. Thetransfections of these vectors were achieved by electroporation using aGene Pulser apparatus (Bio Rad). Briefly, COS cells were suspended inphosphate-buffered saline (PBS) to a cell concentration of 1×10⁷cells/ml, and to 0.8 ml aliquot of the suspension was added 10 μg (pereach plasmid) of DNA. Pulses were applied at 1,500 V and 25 μFcapacitance. After the recovery period of 10 minutes at roomtemperature, the electroporated cells were added to 15 ml of DMEM (GIBCOBRL) containing 5% γ-globulin-free fetal bovine serum. After incubation,a culture supernatant was collected, centrifuged, filtered andaseptically stored. The supernatant was analyzed by ELISA for: (1) theamount of human IgG antibody present in the supernatant and (2) theability of that human IgG to bind to IL-8.

The evaluation of the first version "a" of the reshaped human WS-4 lightchain variable region (RVLa) was conducted by cotransfecting itsexpression vector HEF-RVLa-gκ with the expression vector for chimericWS-4 heavy chain (HEF-chWS4H-gγ1) to produce the antibody "RVLa/chH".

The evaluation of the first version "a" of the reshaped human WS-4 heavychain variable region (RVHa) was conducted by cotransfecting itsexpression vector HEF-RVHa-gγ1 with the expression vector for chimericWS-4 light chain (HEF-chWS4L-gκ) to produce the antibody "chL/RVHa". Asa control, COS cells were also cotransfected with vectors expressingchimeric WS-4 light and heavy chains (HEF-chWS4L-gκ and HEF-chWS4H-gγ1,respectively) to produce the chimeric antibody "chL/chH". Data usingunpurified COS cell supernatants showed that version "a" of the reshapedhuman WS-4 light chain (RVLa) was equivalent to chimeric WS-4 lightchain in assays for binding to IL-8. Version "a" of the reshaped humanWS-4 heavy chain (RVHa), however, virtually abolished binding to IL-8(FIG. 4). Because the version "a" of the reshaped human WS-4 heavy chain(RVHa) failed to bind to IL-8, other versions were constructed accordingto the method described above.

For the version "a", chimeric WS-4 light chain and RVLa were used forevaluating the reshaped human WS-4 heavy chain variable region versions"a" to "h". The evaluation for the eight versions of reshaped human WS-4heavy chain variable regions was conducted by cotransfecting each vectorexpressing reshaped human WS-4 heavy chain (HEF-RVHa-gγ1, HEF-RVHb-gγ1,HEF-RVHc-gγ1, HEF-RVHd-gγ1, HEF-RVHe-gγ1, HEF-RVHf-gγ1, HEF-RVHg-gγ1 orHEF-RVHh-gγ1) with the vector expressing the version "a" of the reshapedhuman WS-4 light chain (HEF-RVLa-gκ) to produce reshaped humanantibodies (RVLa/RVHa, RVLa/RVHb, RVLa/RVHc, RVLa/RVHd, RVLa/RVHe,RVLa/RVHf, RVLa/RVHg and RVLa/RVHh). Cells were also cotransfected withvectors expressing chimeric WS-4 light and heavy chains (HEF-chWS4L-gκand HEF-chWS4H-gγ1, respectively).

Data using unpurified COS cell supernatants showed that bindingactivities to IL-8 of the reshaped human antibodies RVLa/RVHb,RVLa/RVHd, RVLa/RVHe, RVLa/RVHf, RVLa/RVHg and RVLa/RVHh were comparableto that of the chimeric antibody chL/chH. With regard to the productionof antibody, RVLa/RVHg was produced most efficiently among theseantibodies. Consequently, the pairing of version "a" of reshaped humanWS-4 light chain and version "g" of the reshaped human WS-4 heavy chainwas equivalent to the chimeric WS-4 antibody (chL/chH) both in bindingto IL-8 and in production (FIG. 5).

Next, the evaluation of the second version "b" of the reshaped humanWS-4 light chain variable region (RVLb) was conducted by cotransfectingof its expression vector HEF-RVLb-gκ with each expression vector toproduce the reshaped human WS-4 heavy chain as described above. Datausing unpurified COS cell supernatants showed that only pairing ofversion "b" of the reshaped human WS-4 light chain and version "g" ofthe reshaped human WS-4 heavy chain was equivalent to chimeric WS-4antibody (chL/chH) both in binding to IL-8 and in production thereof(FIG. 6).

To confirm these data, chimeric and reshaped human WS-4 antibodies wereconcentrated and purified from COS cell supernatants using Protein A.Namely the culture media from COS cells was concentrated using a 30 kdcut-off ultrafiltration device (Amicon). The concentrated media waspurified using Protein A agarose (Affi-Gel Protein A MAPSII kit,BioRad). Briefly, the concentrated media was applied to a Protein Aagarose column that was equilibrated with five bed volumes of bindingbuffer. The column was washed with 15 bed volumes of the binding buffer,followed by 5 bed volumes of the elution buffer. The eluate wasconcentrated and the buffer changed to PBS using a microconcentrator(Centricon 30, Amicon). The purified antibodies were used for furtheranalysis.

The analysis of purified samples of chimeric WS-4 antibody, and reshapedhuman WS-4 antibodies with versions "a" or "b" of the light chainvariable region and version "g" of the reshaped human heavy chainvariable region, RVLa/RVHg and RVLb/RVHg, was carried out. The bindingof each of the reshaped human antibodies RVLa/RVHg and RVLb/RVHg to IL-8is equivalent to the chimeric WS-4 antibody (FIG. 7).

EXAMPLE 7 Neutralizing Activity

The neutralizing activity of the various forms of humanized anti-IL-8antibody can be assessed in the assays described below for inhibition ofIL-8 receptor binding or inhibition of IL-8-mediated chemotaxis. Bothassays utilize neutrophils. The neutrophils were prepared as follows:About 100 ml of heparinized venous blood was layered onto a Mono-Polyresolving solution (ICN) and centrifuged according to the manufacturer'sinstructions. The neutrophil layer was harvested and washed withRPMI-1640 medium containing 1% BSA. Contaminating red blood cells werelysed by addition of 150 mM ammonium chloride. The suspension wascentrifuged and the pellet containing neutrophils was washed andresuspended at the desired cell density (2×10⁷ cells/ml) in RPMI-1640medium containing 1% BSA. Neutrophils constituted more than 95% of thesuspended cells as determined by the Cytospin assay (Shandon) and by theDiff-Quik staining method (Green Cross Corp.).

For the receptor binding inhibition assay, the neutrophil suspension wascentrifuged and resuspended at a concentration of 2×10⁷ cells/ml inbinding buffer (D-PBS containing 1% BSA and 0.1% sodium azide). In orderto block F_(c) receptors on the neutrophils, another chimeric antibody,SK-2 (PCT application PCT/JP94/00859), was added to a finalconcentration of 50 μg/ml and IL-6 was added to a final concentration of40 ng/ml. The resulting mixture was incubated on ice for 30 min. Thechimeric SK-2 antibody has the same F_(c) region as the humanizedantibody of the present invention and binds human IL-6 forming an immunecomplex.

Then, ¹²⁵ I-labeled IL-8 (74 TBq/mM; Amersham) was admixed withunlabeled IL-8 (Amersham) at an optimal final concentration of 4 ng/mlin binding buffer. WS-4 mouse antibody, chimeric antibody or reshapedhuman antibodies were serially diluted into the binding buffer. Then 50μl each of the IL-8 solution and the antibody solution were mixed andpreincubated on ice for 30 min. After the preincubation, 100 μl of theneutrophil suspension were added and additionally incubated on ice for 1hr, with agitation every 15 min. Following the incubation, the cellsuspension is layered onto a 200 μl, 20% sucrose cushion, centrifugedand frozen. The frozen cell pellets were then cut, and the radioactivitytherein was counted using a γ-counter (Aloka) to measure thecell-associated radiolabeled ligand. The results are shown in FIG. 8.

FIG. 8 shows that the inhibition that results from ligand/receptorbinding of a transiently-produced chimeric antibody (chL/chH) and byreshaped human antibodies (RVLa/RVHg and RVLb/RVHg) was equivalent tothe mouse WS-4 antibody.

The chemotaxis inhibition assay is conducted in a multiwell chemotaxischamber containing a lower and upper compartment and polycarbonate framefilter with a pore size of 5 μm (NeuroProbe Inc.). WS-4 mouse antibody,chimeric antibody or reshaped human antibodies are serially diluted, andadmixed with 20 ng/mL human IL-8 (Amersham) in RPMI-1640 mediumcontaining 1% BSA. Then, 36 μl of this admixture is aliquoted into thewells in the lower compartment-of the chemotaxis chamber and a framefilter is placed on top of the lower compartment. The entire chamber ispreincubated at 37° C. for 30 min, and then 100 μl of a neutrophilsuspension is added to each well in the upper compartment. The chamberis additionally incubated at 37° C. for 1 hr. Neutrophils which migrateand adhere to the membrane are fixed with methanol and stained withGiemsa stain or with Diff-Quik stain (Green Cross Corp.) according tothe manufacturer's instructions. Then the absorbance of the sample at540 nm wave length is measured in a Microplate Reader Model 3350(BioRad), or the cells are directly observed and counted using a lightmicroscope. Alternatively, neutrophils which migrate and adhere to themembrane are fixed with methanol and stained with mouse antihumanneutrophil antibody (BioMakor ™bm) as a primary antibody and FITC(fluorescein isothiocyanate)-conjugated antimouse IgG antibody (BioMakor™bm) as a secondary antibody according to the manufacturer'sinstructions. The fluorescein intensity is then measured in a LeitzPATIMED MPV-MT2 machine (Leica). In all of the above procedures formeasuring chemotaxis, the chemotactic index is defined as the ratio ofthe intensity, absorbance, or direct cell count representing the numberof cells which migrate and adhere to the membrane in the absence ofIL-8, to those cells which migrate and adhere to the membrane in thepresence of IL-8.

Industrial Applicability

The present invention provides a reshaped human antibody to human IL-8,comprising a human antibody wherein the CDRs of the human variableregions are replaced by the CDRs of mouse monoclonal antibodyimmunoreactive with human IL-8. Since a major portion of the reshapedantibody is derived from human immunoglobulin sequences, the presentreshaped human antibody is less immunogenic in humans than antibodiesraised in nonhuman subjects, and is therefore promising for therapeuticuses in humans.

    ______________________________________                                        Reference to Deposited Microorganisms                                         under Rule 13-2 of Budapest Treaty:                                           Identitication of                                                                              Deposition   Deposition                                      Microorganism    Number       Date                                            ______________________________________                                        E. coli DH5α (HEF-chWS4L-gκ)                                                       FERM BP 4739 July 12, 1994                                   E. coli JM109 (HEF-chWS4H-gγ1)                                                           FERM BP 4740 July 12, 1994                                   E. coli DH5α (HEF-RVLa-gκ)                                                         FERM BP 4738 July 12, 1994                                   E. coli JM109 (HEF-RVHg-gγ1)                                                             FERM BP 4741 July 12, 1994                                   ______________________________________                                    

All of the deposited microorganisms have been deposited at the NationalInstitute of Bioscience and Human Technology agency of IndustrialScience and Technology, 1-3, Higashi 1-chome, Tsukuba-Shi, Ibaraki,Japan 305.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 77                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #    40            TTGC CTGTTAGGCT GTTGGTGCTG                                 - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #    39            WCAG ACACACTCCT GYTATGGGT                                  - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #    40            GTGC TCACTCAGGT CCTGGSGTTG                                 - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 43 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 # 43               RCCC CTGCTCAGWT TYTTGGMWTC TTG                             - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #    40            TTWC AGGTGCAGAT TWTCAGCTTC                                 - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #      37          TKCY YTGYTSAGYT YCTGRGG                                    - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 41 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 #   41             WTCA AGATGGAGTC ACAKWYYCWG G                               - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 41 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 #   41             GGAY CTKTTTYCMM TTTTTCAATT G                               - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 35 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 #       35         TCCW CASCTCAGTT CCTTG                                      - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                #      37          ATAT GTTTGTTGTC TATTTCT                                    - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 38 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                #     38           GCCC CAGCTCAGCT TCTCTTCC                                   - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                #             27   GTGG GAAGATG                                               - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                #      37          TGCA GCTGGGTCAT STTCTTC                                    - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 36 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                #       36         TGGA GCTRTATCAT SYTCTT                                     - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                #      37          WTGT GGTTAAACTG GGTTTTT                                    - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 35 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                #       35         TTTG GGYTCAGCTT GRTTT                                      - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                #    40            TCCA GGCTCAATTT AGTTTTCCTT                                 - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                #ID NO:18:(xi) SEQUENCE DESCRIPTION: SEQ                                      #      37          GTCY TRGSGCTRCT CTTCTGC                                    - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 36 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                #       36         TGGA GCKGGRTCTT TMTCTT                                     - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 33 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                #         33       GTGC TGATTCTTTT GTG                                        - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                #    40            TGGG TGTGGAMCTT GCTATTCCTG                                 - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                #      37          AGAC TTACATTCTC ATTCCTG                                    - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 38 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                #     38           TTTG GGCTGATTTT TTTTATTG                                   - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                #      37          GTGT TAAGTCTTCT GTACCTG                                    - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 28 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                #             28   GATA GACAGATG                                              - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 382 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..381                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 61                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                - ATG AGT GTG CTC ACT CAG GTC CTG GGG TTG CT - #G CTG CTG TGG CTT ACA           48                                                                          Met Ser Val Leu Thr Gln Val Leu Gly Leu Le - #u Leu Leu Trp Leu Thr           #-510                                                                         - GGT GCC AGA TGT GAC ATC CAG ATG ACT CAG TC - #T CCA GCC TCC CTA TCT           96                                                                          Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Se - #r Pro Ala Ser Leu Ser           #               10                                                            - GCA TCT GTG GGA GAA ACT GTC ACC ATC ACA TG - #T CGA GCA AGT GAG ATT          144                                                                          Ala Ser Val Gly Glu Thr Val Thr Ile Thr Cy - #s Arg Ala Ser Glu Ile           #         25                                                                  - ATT TAC AGT TAT TTA GCA TGG TAT CAG CAG AA - #A CAG GGA AAA TCT CCT          192                                                                          Ile Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Ly - #s Gln Gly Lys Ser Pro           #     40                                                                      - CAG CTC CTG GTC TAT AAT GCA AAA ACC TTA GC - #A GAT GGT GTG TCA TCA          240                                                                          Gln Leu Leu Val Tyr Asn Ala Lys Thr Leu Al - #a Asp Gly Val Ser Ser           # 60                                                                          - AGG TTC AGT GGC AGT GGA TCA GGC ACA CAG TT - #T TCT CTG CGG ATC AGC          288                                                                          Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Ph - #e Ser Leu Arg Ile Ser           #                 75                                                          - AGC CTG CAG CCT GAA GAT TTT GGG AGT TAT TA - #C TGT CAA CAT CAT TTT          336                                                                          Ser Leu Gln Pro Glu Asp Phe Gly Ser Tyr Ty - #r Cys Gln His His Phe           #             90                                                              - GGT TTT CCT CGG ACG TTC GGT GGA GGC ACC AA - #G CTG GAA CTC AAA              38 - #1                                                                      Gly Phe Pro Arg Thr Phe Gly Gly Gly Thr Ly - #s Leu Glu Leu Lys               #        105                                                                  #              382                                                            - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 127 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                - Met Ser Val Leu Thr Gln Val Leu Gly Leu Le - #u Leu Leu Trp Leu Thr         #-510                                                                         - Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Se - #r Pro Ala Ser Leu Ser         #               10                                                            - Ala Ser Val Gly Glu Thr Val Thr Ile Thr Cy - #s Arg Ala Ser Glu Ile         #         25                                                                  - Ile Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Ly - #s Gln Gly Lys Ser Pro         #     40                                                                      - Gln Leu Leu Val Tyr Asn Ala Lys Thr Leu Al - #a Asp Gly Val Ser Ser         # 60                                                                          - Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Ph - #e Ser Leu Arg Ile Ser         #                 75                                                          - Ser Leu Gln Pro Glu Asp Phe Gly Ser Tyr Ty - #r Cys Gln His His Phe         #             90                                                              - Gly Phe Pro Arg Thr Phe Gly Gly Gly Thr Ly - #s Leu Glu Leu Lys             #        105                                                                  - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 424 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..423                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 58                                                    #ID NO:28:(xi) SEQUENCE DESCRIPTION: SEQ                                      - ATG AAG TTG TGG TTA AAC TGG GTT TTT CTT GT - #G ACA CTT TTA AAT GGT           48                                                                          Met Lys Leu Trp Leu Asn Trp Val Phe Leu Va - #l Thr Leu Leu Asn Gly           - ATC CAG TGT GAG GTG AAA CTG GTG GAG TCT GG - #A GGA GGC TTG ATA CAG           96                                                                          Ile Gln Cys Glu Val Lys Leu Val Glu Ser Gl - #y Gly Gly Leu Ile Gln           #           10                                                                - CCT GGG GAT TCT CTG AGA CTC TCC TGT GTA AC - #C TCT GGG TTC ACC TTC          144                                                                          Pro Gly Asp Ser Leu Arg Leu Ser Cys Val Th - #r Ser Gly Phe Thr Phe           #     25                                                                      - AGT GAT TAC TAC CTG AGC TGG GTC CGC CAG CC - #T CCA GGA AAG GCA CTT          192                                                                          Ser Asp Tyr Tyr Leu Ser Trp Val Arg Gln Pr - #o Pro Gly Lys Ala Leu           # 45                                                                          - GAG TGG GTG GGT CTC ATT AGA AAC AAA GCC AA - #T GGT TAC ACA AGA GAG          240                                                                          Glu Trp Val Gly Leu Ile Arg Asn Lys Ala As - #n Gly Tyr Thr Arg Glu           #                 60                                                          - TAC AGT GCA TCT GTG AAG GGT CGG TTC ACC AT - #C TCC AGA GAT GAT TCC          288                                                                          Tyr Ser Ala Ser Val Lys Gly Arg Phe Thr Il - #e Ser Arg Asp Asp Ser           #             75                                                              - CAA AGC ATC CTC TAT CTT CAA ATG AAC ACC CT - #G AGA GGT GAG GAC AGT          336                                                                          Gln Ser Ile Leu Tyr Leu Gln Met Asn Thr Le - #u Arg Gly Glu Asp Ser           #         90                                                                  - GCC ACT TAT TAC TGT GCA CGA GAG AAC TAT AG - #G TAC GAC GTA GAG CTT          384                                                                          Ala Thr Tyr Tyr Cys Ala Arg Glu Asn Tyr Ar - #g Tyr Asp Val Glu Leu           #    105                                                                      #   424AC TGG GGC CAA GGG ACT CTG GTC ACT GT - #C TCT GCA G                   Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Va - #l Ser Ala                       110                 1 - #15                 1 - #20                           - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 141 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                - Met Lys Leu Trp Leu Asn Trp Val Phe Leu Va - #l Thr Leu Leu Asn Gly         5                                                                             - Ile Gln Cys Glu Val Lys Leu Val Glu Ser Gl - #y Gly Gly Leu Ile Gln         #           10                                                                - Pro Gly Asp Ser Leu Arg Leu Ser Cys Val Th - #r Ser Gly Phe Thr Phe         #     25                                                                      - Ser Asp Tyr Tyr Leu Ser Trp Val Arg Gln Pr - #o Pro Gly Lys Ala Leu         # 45                                                                          - Glu Trp Val Gly Leu Ile Arg Asn Lys Ala As - #n Gly Tyr Thr Arg Glu         #                 60                                                          - Tyr Ser Ala Ser Val Lys Gly Arg Phe Thr Il - #e Ser Arg Asp Asp Ser         #             75                                                              - Gln Ser Ile Leu Tyr Leu Gln Met Asn Thr Le - #u Arg Gly Glu Asp Ser         #         90                                                                  - Ala Thr Tyr Tyr Cys Ala Arg Glu Asn Tyr Ar - #g Tyr Asp Val Glu Leu         #    105                                                                      - Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Va - #l Ser Ala                     110                 1 - #15                 1 - #20                           - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 34 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                #        34        GAGT GTGCTCACTC AGGT                                       - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                #      37          GAAG TTGTGGTTAA ACTGGGT                                    - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                #      37          TTTG AGTTCCAGCT TGGTGCC                                    - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #33:                           #      37          TGCA GAGACAGTGA CCAGAGT                                    - (2) INFORMATION FOR SEQ ID NO:34:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 137 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                - TAAGCTTCCA CCATGGAGTT TGGGCTGAGC TGGGTTTTCC TTGTTGCTAT TT - #TAAAGGGT         60                                                                          - GTCCAGTGTG AAGTGCAGCT GTTGGAGTCT GGGGGAGGCT TGGTCCAGCC TG - #GGGGTTCT        120                                                                          #  137             C                                                          - (2) INFORMATION FOR SEQ ID NO:35:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 143 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                - GCACTGTACT CTCTTGTGTA ACCATTGGCT TTGTTTCTAA TGAGACCCAC CA - #ACTCTAGC         60                                                                          - CCTTTCCCTT GAGCTTGGCG GACCCAGCTC AGGTAGTAAT CACTGAAGGT GA - #ATCCAGAG        120                                                                          #               143TCAG AGA                                                   - (2) INFORMATION FOR SEQ ID NO:36:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 113 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                - TACACAAGAG AGTACAGTGC ATCTGTGAAG GGCAGACTTA CCATCTCAAG AG - #AAGATTCA         60                                                                          - AAGAACACGC TGTATCTGCA AATGAGCAGC CTGAAAACCG AAGACTTGGC CG - #T               113                                                                          - (2) INFORMATION FOR SEQ ID NO:37:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 117 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                - TCGGATCCAC TCACCTGAGG AGACGGTGAC CAGGGTTCCC TGGCCCCAGT AA - #GCAAGCTC         60                                                                          - TACGTCGTAG CGATAGTTCT CTCTAGCACA GTAATACACG GCCAAGTCTT CG - #GTTTT           117                                                                          - (2) INFORMATION FOR SEQ ID NO:38:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                #ID NO:38:(xi) SEQUENCE DESCRIPTION: SEQ                                      #      37          GGAG TTTGGGCTGA GCTGGGT                                    - (2) INFORMATION FOR SEQ ID NO:39:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                #          31      TGAG GAGACGGTGA C                                          - (2) INFORMATION FOR SEQ ID NO:40:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 424 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..423                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 58                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                - ATG GAG TTT GGG CTG AGC TGG GTT TTC CTT GT - #T GCT ATT TTA AAG GGT           48                                                                          Met Glu Phe Gly Leu Ser Trp Val Phe Leu Va - #l Ala Ile Leu Lys Gly           5                                                                             - GTC CAG TGT GAA GTG CAG CTG TTG GAG TCT GG - #G GGA GGC TTG GTC CAG           96                                                                          Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gl - #y Gly Gly Leu Val Gln           #           10                                                                - CCT GGG GGT TCT CTG AGA CTC TCA TGT GCT GC - #C TCT GGA TTC ACC TTC          144                                                                          Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Al - #a Ser Gly Phe Thr Phe           #     25                                                                      - AGT GAT TAC TAC CTG AGC TGG GTC CGC CAA GC - #T CAA GGG AAA GGG CTA          192                                                                          Ser Asp Tyr Tyr Leu Ser Trp Val Arg Gln Al - #a Gln Gly Lys Gly Leu           # 45                                                                          - GAG TTG GTG GGT CTC ATT AGA AAC AAA GCC AA - #T GGT TAC ACA AGA GAG          240                                                                          Glu Leu Val Gly Leu Ile Arg Asn Lys Ala As - #n Gly Tyr Thr Arg Glu           #                 60                                                          - TAC AGT GCA TCT GTG AAG GGC AGA CTT ACC AT - #C TCA AGA GAA GAT TCA          288                                                                          Tyr Ser Ala Ser Val Lys Gly Arg Leu Thr Il - #e Ser Arg Glu Asp Ser           #             75                                                              - AAG AAC ACG CTG TAT CTG CAA ATG AGC AGC CT - #G AAA ACC GAA GAC TTG          336                                                                          Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Le - #u Lys Thr Glu Asp Leu           #         90                                                                  - GCC GTG TAT TAC TGT GCT AGA GAG AAC TAT CG - #C TAC GAC GTA GAG CTT          384                                                                          Ala Val Tyr Tyr Cys Ala Arg Glu Asn Tyr Ar - #g Tyr Asp Val Glu Leu           #    105                                                                      #   424AC TGG GGC CAG GGA ACC CTG GTC ACC GT - #C TCC TCA G                   Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Va - #l Ser Ser                       110                 1 - #15                 1 - #20                           - (2) INFORMATION FOR SEQ ID NO:41:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 141 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                - Met Glu Phe Gly Leu Ser Trp Val Phe Leu Va - #l Ala Ile Leu Lys Gly         5                                                                             - Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gl - #y Gly Gly Leu Val Gln         #           10                                                                - Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Al - #a Ser Gly Phe Thr Phe         #     25                                                                      - Ser Asp Tyr Tyr Leu Ser Trp Val Arg Gln Al - #a Gln Gly Lys Gly Leu         # 45                                                                          - Glu Leu Val Gly Leu Ile Arg Asn Lys Ala As - #n Gly Tyr Thr Arg Glu         #                 60                                                          - Tyr Ser Ala Ser Val Lys Gly Arg Leu Thr Il - #e Ser Arg Glu Asp Ser         #             75                                                              - Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Le - #u Lys Thr Glu Asp Leu         #         90                                                                  - Ala Val Tyr Tyr Cys Ala Arg Glu Asn Tyr Ar - #g Tyr Asp Val Glu Leu         #    105                                                                      - Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Va - #l Ser Ser                     110                 1 - #15                 1 - #20                           - (2) INFORMATION FOR SEQ ID NO:42:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 34 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                #        34        TCTC ATTAGAAACA AAGC                                       - (2) INFORMATION FOR SEQ ID NO:43:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 36 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                #       36         AGCC CTTTCCCTTG AGCTTG                                     - (2) INFORMATION FOR SEQ ID NO:44:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 424 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..423                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 58                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                - ATG GAG TTT GGG CTG AGC TGG GTT TTC CTT GT - #T GCT ATT TTA AAG GGT           48                                                                          Met Glu Phe Gly Leu Ser Trp Val Phe Leu Va - #l Ala Ile Leu Lys Gly           5                                                                             - GTC CAG TGT GAA GTG CAG CTG TTG GAG TCT GG - #G GGA GGC TTG GTC CAG           96                                                                          Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gl - #y Gly Gly Leu Val Gln           #           10                                                                - CCT GGG GGT TCT CTG AGA CTC TCA TGT GCT GC - #C TCT GGA TTC ACC TTC          144                                                                          Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Al - #a Ser Gly Phe Thr Phe           #     25                                                                      - AGT GAT TAC TAC CTG AGC TGG GTC CGC CAA GC - #T CAA GGG AAA GGG CTA          192                                                                          Ser Asp Tyr Tyr Leu Ser Trp Val Arg Gln Al - #a Gln Gly Lys Gly Leu           # 45                                                                          - GAG TGG GTG GGT CTC ATT AGA AAC AAA GCC AA - #T GGT TAC ACA AGA GAG          240                                                                          Glu Trp Val Gly Leu Ile Arg Asn Lys Ala As - #n Gly Tyr Thr Arg Glu           #                 60                                                          - TAC AGT GCA TCT GTG AAG GGC AGA CTT ACC AT - #C TCA AGA GAA GAT TCA          288                                                                          Tyr Ser Ala Ser Val Lys Gly Arg Leu Thr Il - #e Ser Arg Glu Asp Ser           #             75                                                              - AAG AAC ACG CTG TAT CTG CAA ATG AGC AGC CT - #G AAA ACC GAA GAC TTG          336                                                                          Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Le - #u Lys Thr Glu Asp Leu           #         90                                                                  - GCC GTG TAT TAC TGT GCT AGA GAG AAC TAT CG - #C TAC GAC GTA GAG CTT          384                                                                          Ala Val Tyr Tyr Cys Ala Arg Glu Asn Tyr Ar - #g Tyr Asp Val Glu Leu           #    105                                                                      #   424AC TGG GGC CAG GGA ACC CTG GTC ACC GT - #C TCC TCA G                   Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Va - #l Ser Ser                       110                 1 - #15                 1 - #20                           - (2) INFORMATION FOR SEQ ID NO:45:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 141 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                - Met Glu Phe Gly Leu Ser Trp Val Phe Leu Va - #l Ala Ile Leu Lys Gly         5                                                                             - Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gl - #y Gly Gly Leu Val Gln         #           10                                                                - Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Al - #a Ser Gly Phe Thr Phe         #     25                                                                      - Ser Asp Tyr Tyr Leu Ser Trp Val Arg Gln Al - #a Gln Gly Lys Gly Leu         # 45                                                                          - Glu Trp Val Gly Leu Ile Arg Asn Lys Ala As - #n Gly Tyr Thr Arg Glu         #                 60                                                          - Tyr Ser Ala Ser Val Lys Gly Arg Leu Thr Il - #e Ser Arg Glu Asp Ser         #             75                                                              - Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Le - #u Lys Thr Glu Asp Leu         #         90                                                                  - Ala Val Tyr Tyr Cys Ala Arg Glu Asn Tyr Ar - #g Tyr Asp Val Glu Leu         #    105                                                                      - Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Va - #l Ser Ser                     110                 1 - #15                 1 - #20                           - (2) INFORMATION FOR SEQ ID NO:46:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                #          32      CAGG GAAAGGGCTA GA                                         - (2) INFORMATION FOR SEQ ID NO:47:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                #          32      GAGC TTGGCGGACC CA                                         - (2) INFORMATION FOR SEQ ID NO:48:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 424 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..423                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 58                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                - ATG GAG TTT GGG CTG AGC TGG GTT TTC CTT GT - #T GCT ATT TTA AAG GGT           48                                                                          Met Glu Phe Gly Leu Ser Trp Val Phe Leu Va - #l Ala Ile Leu Lys Gly           5                                                                             - GTC CAG TGT GAA GTG CAG CTG TTG GAG TCT GG - #G GGA GGC TTG GTC CAG           96                                                                          Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gl - #y Gly Gly Leu Val Gln           #           10                                                                - CCT GGG GGT TCT CTG AGA CTC TCA TGT GCT GC - #C TCT GGA TTC ACC TTC          144                                                                          Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Al - #a Ser Gly Phe Thr Phe           #     25                                                                      - AGT GAT TAC TAC CTG AGC TGG GTC CGC CAA GC - #T CCA GGG AAA GGG CTA          192                                                                          Ser Asp Tyr Tyr Leu Ser Trp Val Arg Gln Al - #a Pro Gly Lys Gly Leu           # 45                                                                          - GAG TTG GTG GGT CTC ATT AGA AAC AAA GCC AA - #T GGT TAC ACA AGA GAG          240                                                                          Glu Leu Val Gly Leu Ile Arg Asn Lys Ala As - #n Gly Tyr Thr Arg Glu           #                 60                                                          - TAC AGT GCA TCT GTG AAG GGC AGA CTT ACC AT - #C TCA AGA GAA GAT TCA          288                                                                          Tyr Ser Ala Ser Val Lys Gly Arg Leu Thr Il - #e Ser Arg Glu Asp Ser           #             75                                                              - AAG AAC ACG CTG TAT CTG CAA ATG AGC AGC CT - #G AAA ACC GAA GAC TTG          336                                                                          Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Le - #u Lys Thr Glu Asp Leu           #         90                                                                  - GCC GTG TAT TAC TGT GCT AGA GAG AAC TAT CG - #C TAC GAC GTA GAG CTT          384                                                                          Ala Val Tyr Tyr Cys Ala Arg Glu Asn Tyr Ar - #g Tyr Asp Val Glu Leu           #    105                                                                      #   424AC TGG GGC CAG GGA ACC CTG GTC ACC GT - #C TCC TCA G                   Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Va - #l Ser Ser                       110                 1 - #15                 1 - #20                           - (2) INFORMATION FOR SEQ ID NO:49:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 141 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                - Met Glu Phe Gly Leu Ser Trp Val Phe Leu Va - #l Ala Ile Leu Lys Gly         5                                                                             - Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gl - #y Gly Gly Leu Val Gln         #           10                                                                - Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Al - #a Ser Gly Phe Thr Phe         #     25                                                                      - Ser Asp Tyr Tyr Leu Ser Trp Val Arg Gln Al - #a Pro Gly Lys Gly Leu         # 45                                                                          - Glu Leu Val Gly Leu Ile Arg Asn Lys Ala As - #n Gly Tyr Thr Arg Glu         #                 60                                                          - Tyr Ser Ala Ser Val Lys Gly Arg Leu Thr Il - #e Ser Arg Glu Asp Ser         #             75                                                              - Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Le - #u Lys Thr Glu Asp Leu         #         90                                                                  - Ala Val Tyr Tyr Cys Ala Arg Glu Asn Tyr Ar - #g Tyr Asp Val Glu Leu         #    105                                                                      - Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Va - #l Ser Ser                     110                 1 - #15                 1 - #20                           - (2) INFORMATION FOR SEQ ID NO:50:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 424 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..423                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 58                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                - ATG GAG TTT GGG CTG AGC TGG GTT TTC CTT GT - #T GCT ATT TTA AAG GGT           48                                                                          Met Glu Phe Gly Leu Ser Trp Val Phe Leu Va - #l Ala Ile Leu Lys Gly           5                                                                             - GTC CAG TGT GAA GTG CAG CTG TTG GAG TCT GG - #G GGA GGC TTG GTC CAG           96                                                                          Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gl - #y Gly Gly Leu Val Gln           #           10                                                                - CCT GGG GGT TCT CTG AGA CTC TCA TGT GCT GC - #C TCT GGA TTC ACC TTC          144                                                                          Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Al - #a Ser Gly Phe Thr Phe           #     25                                                                      - AGT GAT TAC TAC CTG AGC TGG GTC CGC CAA GC - #T CCA GGG AAA GGG CTA          192                                                                          Ser Asp Tyr Tyr Leu Ser Trp Val Arg Gln Al - #a Pro Gly Lys Gly Leu           # 45                                                                          - GAG TGG GTG GGT CTC ATT AGA AAC AAA GCC AA - #T GGT TAC ACA AGA GAG          240                                                                          Glu Trp Val Gly Leu Ile Arg Asn Lys Ala As - #n Gly Tyr Thr Arg Glu           #                 60                                                          - TAC AGT GCA TCT GTG AAG GGC AGA CTT ACC AT - #C TCA AGA GAA GAT TCA          288                                                                          Tyr Ser Ala Ser Val Lys Gly Arg Leu Thr Il - #e Ser Arg Glu Asp Ser           #             75                                                              - AAG AAC ACG CTG TAT CTG CAA ATG AGC AGC CT - #G AAA ACC GAA GAC TTG          336                                                                          Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Le - #u Lys Thr Glu Asp Leu           #         90                                                                  - GCC GTG TAT TAC TGT GCT AGA GAG AAC TAT CG - #C TAC GAC GTA GAG CTT          384                                                                          Ala Val Tyr Tyr Cys Ala Arg Glu Asn Tyr Ar - #g Tyr Asp Val Glu Leu           #    105                                                                      #   424AC TGG GGC CAG GGA ACC CTG GTC ACC GT - #C TCC TCA G                   Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Va - #l Ser Ser                       110                 1 - #15                 1 - #20                           - (2) INFORMATION FOR SEQ ID NO:51:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 141 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                - Met Glu Phe Gly Leu Ser Trp Val Phe Leu Va - #l Ala Ile Leu Lys Gly         5                                                                             - Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gl - #y Gly Gly Leu Val Gln         #           10                                                                - Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Al - #a Ser Gly Phe Thr Phe         #     25                                                                      - Ser Asp Tyr Tyr Leu Ser Trp Val Arg Gln Al - #a Pro Gly Lys Gly Leu         # 45                                                                          - Glu Trp Val Gly Leu Ile Arg Asn Lys Ala As - #n Gly Tyr Thr Arg Glu         #                 60                                                          - Tyr Ser Ala Ser Val Lys Gly Arg Leu Thr Il - #e Ser Arg Glu Asp Ser         #             75                                                              - Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Le - #u Lys Thr Glu Asp Leu         #         90                                                                  - Ala Val Tyr Tyr Cys Ala Arg Glu Asn Tyr Ar - #g Tyr Asp Val Glu Leu         #    105                                                                      - Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Va - #l Ser Ser                     110                 1 - #15                 1 - #20                           - (2) INFORMATION FOR SEQ ID NO:52:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                #              26  CAGG GAAAGG                                                - (2) INFORMATION FOR SEQ ID NO:53:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                #              26  GGCG GACCCA                                                - (2) INFORMATION FOR SEQ ID NO:54:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 424 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..423                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 58                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                - ATG GAG TTT GGG CTG AGC TGG GTT TTC CTT GT - #T GCT ATT TTA AAG GGT           48                                                                          Met Glu Phe Gly Leu Ser Trp Val Phe Leu Va - #l Ala Ile Leu Lys Gly           5                                                                             - GTC CAG TGT GAA GTG CAG CTG TTG GAG TCT GG - #G GGA GGC TTG GTC CAG           96                                                                          Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gl - #y Gly Gly Leu Val Gln           #           10                                                                - CCT GGG GGT TCT CTG AGA CTC TCA TGT GCT GC - #C TCT GGA TTC ACC TTC          144                                                                          Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Al - #a Ser Gly Phe Thr Phe           #     25                                                                      - AGT GAT TAC TAC CTG AGC TGG GTC CGC CAA CC - #T CCA GGG AAA GGG CTA          192                                                                          Ser Asp Tyr Tyr Leu Ser Trp Val Arg Gln Pr - #o Pro Gly Lys Gly Leu           # 45                                                                          - GAG TGG GTG GGT CTC ATT AGA AAC AAA GCC AA - #T GGT TAC ACA AGA GAG          240                                                                          Glu Trp Val Gly Leu Ile Arg Asn Lys Ala As - #n Gly Tyr Thr Arg Glu           #                 60                                                          - TAC AGT GCA TCT GTG AAG GGC AGA CTT ACC AT - #C TCA AGA GAA GAT TCA          288                                                                          Tyr Ser Ala Ser Val Lys Gly Arg Leu Thr Il - #e Ser Arg Glu Asp Ser           #             75                                                              - AAG AAC ACG CTG TAT CTG CAA ATG AGC AGC CT - #G AAA ACC GAA GAC TTG          336                                                                          Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Le - #u Lys Thr Glu Asp Leu           #         90                                                                  - GCC GTG TAT TAC TGT GCT AGA GAG AAC TAT CG - #C TAC GAC GTA GAG CTT          384                                                                          Ala Val Tyr Tyr Cys Ala Arg Glu Asn Tyr Ar - #g Tyr Asp Val Glu Leu           #    105                                                                      #   424AC TGG GGC CAG GGA ACC CTG GTC ACC GT - #C TCC TCA G                   Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Va - #l Ser Ser                       110                 1 - #15                 1 - #20                           - (2) INFORMATION FOR SEQ ID NO:55:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 141 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                - Met Glu Phe Gly Leu Ser Trp Val Phe Leu Va - #l Ala Ile Leu Lys Gly         5                                                                             - Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gl - #y Gly Gly Leu Val Gln         #           10                                                                - Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Al - #a Ser Gly Phe Thr Phe         #     25                                                                      - Ser Asp Tyr Tyr Leu Ser Trp Val Arg Gln Pr - #o Pro Gly Lys Gly Leu         # 45                                                                          - Glu Trp Val Gly Leu Ile Arg Asn Lys Ala As - #n Gly Tyr Thr Arg Glu         #                 60                                                          - Tyr Ser Ala Ser Val Lys Gly Arg Leu Thr Il - #e Ser Arg Glu Asp Ser         #             75                                                              - Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Le - #u Lys Thr Glu Asp Leu         #         90                                                                  - Ala Val Tyr Tyr Cys Ala Arg Glu Asn Tyr Ar - #g Tyr Asp Val Glu Leu         #    105                                                                      - Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Va - #l Ser Ser                     110                 1 - #15                 1 - #20                           - (2) INFORMATION FOR SEQ ID NO:56:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 29 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                #            29    CGCT AGAGTGGGT                                             - (2) INFORMATION FOR SEQ ID NO:57:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 29 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                #            29    TCCC TGGAGCTTG                                             - (2) INFORMATION FOR SEQ ID NO:58:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 424 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..423                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 58                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                - ATG GAG TTT GGG CTG AGC TGG GTT TTC CTT GT - #T GCT ATT TTA AAG GGT           48                                                                          Met Glu Phe Gly Leu Ser Trp Val Phe Leu Va - #l Ala Ile Leu Lys Gly           5                                                                             - GTC CAG TGT GAA GTG CAG CTG TTG GAG TCT GG - #G GGA GGC TTG GTC CAG           96                                                                          Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gl - #y Gly Gly Leu Val Gln           #           10                                                                - CCT GGG GGT TCT CTG AGA CTC TCA TGT GCT GC - #C TCT GGA TTC ACC TTC          144                                                                          Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Al - #a Ser Gly Phe Thr Phe           #     25                                                                      - AGT GAT TAC TAC CTG AGC TGG GTC CGC CAA GC - #T CCA GGG AAA GCG CTA          192                                                                          Ser Asp Tyr Tyr Leu Ser Trp Val Arg Gln Al - #a Pro Gly Lys Ala Leu           # 45                                                                          - GAG TGG GTG GGT CTC ATT AGA AAC AAA GCC AA - #T GGT TAC ACA AGA GAG          240                                                                          Glu Trp Val Gly Leu Ile Arg Asn Lys Ala As - #n Gly Tyr Thr Arg Glu           #                 60                                                          - TAC AGT GCA TCT GTG AAG GGC AGA CTT ACC AT - #C TCA AGA GAA GAT TCA          288                                                                          Tyr Ser Ala Ser Val Lys Gly Arg Leu Thr Il - #e Ser Arg Glu Asp Ser           #             75                                                              - AAG AAC ACG CTG TAT CTG CAA ATG AGC AGC CT - #G AAA ACC GAA GAC TTG          336                                                                          Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Le - #u Lys Thr Glu Asp Leu           #         90                                                                  - GCC GTG TAT TAC TGT GCT AGA GAG AAC TAT CG - #C TAC GAC GTA GAG CTT          384                                                                          Ala Val Tyr Tyr Cys Ala Arg Glu Asn Tyr Ar - #g Tyr Asp Val Glu Leu           #    105                                                                      #   424AC TGG GGC CAG GGA ACC CTG GTC ACC GT - #C TCC TCA G                   Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Va - #l Ser Ser                       110                 1 - #15                 1 - #20                           - (2) INFORMATION FOR SEQ ID NO:59:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 141 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                - Met Glu Phe Gly Leu Ser Trp Val Phe Leu Va - #l Ala Ile Leu Lys Gly         5                                                                             - Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gl - #y Gly Gly Leu Val Gln         #           10                                                                - Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Al - #a Ser Gly Phe Thr Phe         #     25                                                                      - Ser Asp Tyr Tyr Leu Ser Trp Val Arg Gln Al - #a Pro Gly Lys Ala Leu         # 45                                                                          - Glu Trp Val Gly Leu Ile Arg Asn Lys Ala As - #n Gly Tyr Thr Arg Glu         #                 60                                                          - Tyr Ser Ala Ser Val Lys Gly Arg Leu Thr Il - #e Ser Arg Glu Asp Ser         #             75                                                              - Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Le - #u Lys Thr Glu Asp Leu         #         90                                                                  - Ala Val Tyr Tyr Cys Ala Arg Glu Asn Tyr Ar - #g Tyr Asp Val Glu Leu         #    105                                                                      - Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Va - #l Ser Ser                     110                 1 - #15                 1 - #20                           - (2) INFORMATION FOR SEQ ID NO:60:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                #                23CCAT CTC                                                   - (2) INFORMATION FOR SEQ ID NO:61:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                #                23CCTT CAC                                                   - (2) INFORMATION FOR SEQ ID NO:62:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 424 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..423                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 58                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                - ATG GAG TTT GGG CTG AGC TGG GTT TTC CTT GT - #T GCT ATT TTA AAG GGT           48                                                                          Met Glu Phe Gly Leu Ser Trp Val Phe Leu Va - #l Ala Ile Leu Lys Gly           5                                                                             - GTC CAG TGT GAA GTG CAG CTG TTG GAG TCT GG - #G GGA GGC TTG GTC CAG           96                                                                          Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gl - #y Gly Gly Leu Val Gln           #           10                                                                - CCT GGG GGT TCT CTG AGA CTC TCA TGT GCT GC - #C TCT GGA TTC ACC TTC          144                                                                          Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Al - #a Ser Gly Phe Thr Phe           #     25                                                                      - AGT GAT TAC TAC CTG AGC TGG GTC CGC CAA GC - #T CCA GGG AAA GGG CTA          192                                                                          Ser Asp Tyr Tyr Leu Ser Trp Val Arg Gln Al - #a Pro Gly Lys Gly Leu           # 45                                                                          - GAG TGG GTG GGT CTC ATT AGA AAC AAA GCC AA - #T GGT TAC ACA AGA GAG          240                                                                          Glu Trp Val Gly Leu Ile Arg Asn Lys Ala As - #n Gly Tyr Thr Arg Glu           #                 60                                                          - TAC AGT GCA TCT GTG AAG GGC AGA TTT ACC AT - #C TCA AGA GAA GAT TCA          288                                                                          Tyr Ser Ala Ser Val Lys Gly Arg Phe Thr Il - #e Ser Arg Glu Asp Ser           #             75                                                              - AAG AAC ACG CTG TAT CTG CAA ATG AGC AGC CT - #G AAA ACC GAA GAC TTG          336                                                                          Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Le - #u Lys Thr Glu Asp Leu           #         90                                                                  - GCC GTG TAT TAC TGT GCT AGA GAG AAC TAT CG - #C TAC GAC GTA GAG CTT          384                                                                          Ala Val Tyr Tyr Cys Ala Arg Glu Asn Tyr Ar - #g Tyr Asp Val Glu Leu           #    105                                                                      #   424AC TGG GGC CAG GGA ACC CTG GTC ACC GT - #C TCC TCA G                   Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Va - #l Ser Ser                       110                 1 - #15                 1 - #20                           - (2) INFORMATION FOR SEQ ID NO:63:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 141 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                - Met Glu Phe Gly Leu Ser Trp Val Phe Leu Va - #l Ala Ile Leu Lys Gly         5                                                                             - Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gl - #y Gly Gly Leu Val Gln         #           10                                                                - Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Al - #a Ser Gly Phe Thr Phe         #     25                                                                      - Ser Asp Tyr Tyr Leu Ser Trp Val Arg Gln Al - #a Pro Gly Lys Gly Leu         # 45                                                                          - Glu Trp Val Gly Leu Ile Arg Asn Lys Ala As - #n Gly Tyr Thr Arg Glu         #                 60                                                          - Tyr Ser Ala Ser Val Lys Gly Arg Phe Thr Il - #e Ser Arg Glu Asp Ser         #             75                                                              - Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Le - #u Lys Thr Glu Asp Leu         #         90                                                                  - Ala Val Tyr Tyr Cys Ala Arg Glu Asn Tyr Ar - #g Tyr Asp Val Glu Leu         #    105                                                                      - Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Va - #l Ser Ser                     110                 1 - #15                 1 - #20                           - (2) INFORMATION FOR SEQ ID NO:64:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 424 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..423                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 58                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                - ATG GAG TTT GGG CTG AGC TGG GTT TTC CTT GT - #T GCT ATT TTA AAG GGT           48                                                                          Met Glu Phe Gly Leu Ser Trp Val Phe Leu Va - #l Ala Ile Leu Lys Gly           5                                                                             - GTC CAG TGT GAA GTG CAG CTG TTG GAG TCT GG - #G GGA GGC TTG GTC CAG           96                                                                          Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gl - #y Gly Gly Leu Val Gln           #           10                                                                - CCT GGG GGT TCT CTG AGA CTC TCA TGT GCT GC - #C TCT GGA TTC ACC TTC          144                                                                          Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Al - #a Ser Gly Phe Thr Phe           #     25                                                                      - AGT GAT TAC TAC CTG AGC TGG GTC CGC CAA GC - #T CAA GGG AAA GGG CTA          192                                                                          Ser Asp Tyr Tyr Leu Ser Trp Val Arg Gln Al - #a Gln Gly Lys Gly Leu           # 45                                                                          - GAG TGG GTG GGT CTC ATT AGA AAC AAA GCC AA - #T GGT TAC ACA AGA GAG          240                                                                          Glu Trp Val Gly Leu Ile Arg Asn Lys Ala As - #n Gly Tyr Thr Arg Glu           #                 60                                                          - TAC AGT GCA TCT GTG AAG GGC AGA TTT ACC AT - #C TCA AGA GAA GAT TCA          288                                                                          Tyr Ser Ala Ser Val Lys Gly Arg Phe Thr Il - #e Ser Arg Glu Asp Ser           #             75                                                              - AAG AAC ACG CTG TAT CTG CAA ATG AGC AGC CT - #G AAA ACC GAA GAC TTG          336                                                                          Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Le - #u Lys Thr Glu Asp Leu           #         90                                                                  - GCC GTG TAT TAC TGT GCT AGA GAG AAC TAT CG - #C TAC GAC GTA GAG CTT          384                                                                          Ala Val Tyr Tyr Cys Ala Arg Glu Asn Tyr Ar - #g Tyr Asp Val Glu Leu           #    105                                                                      #   424AC TGG GGC CAG GGA ACC CTG GTC ACC GT - #C TCC TCA G                   Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Va - #l Ser Ser                       110                 1 - #15                 1 - #20                           - (2) INFORMATION FOR SEQ ID NO:65:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 141 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                - Met Glu Phe Gly Leu Ser Trp Val Phe Leu Va - #l Ala Ile Leu Lys Gly         5                                                                             - Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gl - #y Gly Gly Leu Val Gln         #           10                                                                - Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Al - #a Ser Gly Phe Thr Phe         #     25                                                                      - Ser Asp Tyr Tyr Leu Ser Trp Val Arg Gln Al - #a Gln Gly Lys Gly Leu         # 45                                                                          - Glu Trp Val Gly Leu Ile Arg Asn Lys Ala As - #n Gly Tyr Thr Arg Glu         #                 60                                                          - Tyr Ser Ala Ser Val Lys Gly Arg Phe Thr Il - #e Ser Arg Glu Asp Ser         #             75                                                              - Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Le - #u Lys Thr Glu Asp Leu         #         90                                                                  - Ala Val Tyr Tyr Cys Ala Arg Glu Asn Tyr Ar - #g Tyr Asp Val Glu Leu         #    105                                                                      - Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Va - #l Ser Ser                     110                 1 - #15                 1 - #20                           - (2) INFORMATION FOR SEQ ID NO:66:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 124 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                - TTGAAGCTTC CACCATGGGA TGGAGCTGTA TCATCCTCTT CTTGGTAGCA AC - #AGCTACAG         60                                                                          - GTGTCCACTC CGACATCCAG ATGACCCAGA GCCCAAGCAG CCTGAGCGCC AG - #CGTAGGTG        120                                                                          #            124                                                              - (2) INFORMATION FOR SEQ ID NO:67:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 122 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                - GCATTGTAGA TCAGCAGCTT TGGAGCCTTT CCTGGCTTCT GCTGGTACCA TG - #CTAAATAA         60                                                                          - CTGTAAATAA TCTCGCTTGC TCGACAGGTG ATGGTCACTC TGTCACCTAC GC - #TGGCGCTC        120                                                                          #             122                                                             - (2) INFORMATION FOR SEQ ID NO:68:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 121 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                - AGCTGCTGAT CTACAATGCA AAAACCTTAG CAGATGGAGT GCCAAGCAGA TT - #CAGCGGTA         60                                                                          - GCGGTAGCGG TACCGACTTC ACCTTCACCA TCAGCAGCCT CCAGCCAGAG GA - #CATCGCTA        120                                                                          #              121                                                            - (2) INFORMATION FOR SEQ ID NO:69:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 106 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                - GTAGGATCCA CTCACGTTTG ATTTCGACCT TGGTCCCTTG GCCGAACGTC CG - #AGGAAAAC         60                                                                          #                106TAG TAGGTAGCGA TGTCCTCTGG CTGGAG                          - (2) INFORMATION FOR SEQ ID NO:70:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                                # 20               GGGA                                                       - (2) INFORMATION FOR SEQ ID NO:71:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                                # 20               TTTG                                                       - (2) INFORMATION FOR SEQ ID NO:72:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 379 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..378                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 58                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                                - ATG GGA TGG AGC TGT ATC ATC CTC TTC TTG GT - #A GCA ACA GCT ACA GGT           48                                                                          Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Va - #l Ala Thr Ala Thr Gly           5                                                                             - GTC CAC TCC GAC ATC CAG ATG ACC CAG AGC CC - #A AGC AGC CTG AGC GCC           96                                                                          Val His Ser Asp Ile Gln Met Thr Gln Ser Pr - #o Ser Ser Leu Ser Ala           #           10                                                                - AGC GTA GGT GAC AGA GTG ACC ATC ACC TGT CG - #A GCA AGC GAG ATT ATT          144                                                                          Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ar - #g Ala Ser Glu Ile Ile           #     25                                                                      - TAC AGT TAT TTA GCA TGG TAC CAG CAG AAG CC - #A GGA AAG GCT CCA AAG          192                                                                          Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pr - #o Gly Lys Ala Pro Lys           # 45                                                                          - CTG CTG ATC TAC AAT GCA AAA ACC TTA GCA GA - #T GGA GTG CCA AGC AGA          240                                                                          Leu Leu Ile Tyr Asn Ala Lys Thr Leu Ala As - #p Gly Val Pro Ser Arg           #                 60                                                          - TTC AGC GGT AGC GGT AGC GGT ACC GAC TTC AC - #C TTC ACC ATC AGC AGC          288                                                                          Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Th - #r Phe Thr Ile Ser Ser           #             75                                                              - CTC CAG CCA GAG GAC ATC GCT ACC TAC TAC TG - #C CAA CAT CAT TTT GGT          336                                                                          Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cy - #s Gln His His Phe Gly           #         90                                                                  - TTT CCT CGG ACG TTC GGC CAA GGG ACC AAG GT - #C GAA ATC AAA                 # 378                                                                         Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Va - #l Glu Ile Lys                   #    105                                                                      #              379                                                            - (2) INFORMATION FOR SEQ ID NO:73:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 126 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                                - Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Va - #l Ala Thr Ala Thr Gly         5                                                                             - Val His Ser Asp Ile Gln Met Thr Gln Ser Pr - #o Ser Ser Leu Ser Ala         #           10                                                                - Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ar - #g Ala Ser Glu Ile Ile         #     25                                                                      - Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pr - #o Gly Lys Ala Pro Lys         # 45                                                                          - Leu Leu Ile Tyr Asn Ala Lys Thr Leu Ala As - #p Gly Val Pro Ser Arg         #                 60                                                          - Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Th - #r Phe Thr Ile Ser Ser         #             75                                                              - Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cy - #s Gln His His Phe Gly         #         90                                                                  - Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Va - #l Glu Ile Lys                 #    105                                                                      - (2) INFORMATION FOR SEQ ID NO:74:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 38 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                                #     38           ACTA CACCTTCACC ATCAGCAG                                   - (2) INFORMATION FOR SEQ ID NO:75:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 38 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                                #     38           TGTA GTCGGTACCG CTACCGCT                                   - (2) INFORMATION FOR SEQ ID NO:76:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 379 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..378                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 58                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                                - ATG GGA TGG AGC TGT ATC ATC CTC TTC TTG GT - #A GCA ACA GCT ACA GGT           48                                                                          Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Va - #l Ala Thr Ala Thr Gly           5                                                                             - GTC CAC TCC GAC ATC CAG ATG ACC CAG AGC CC - #A AGC AGC CTG AGC GCC           96                                                                          Val His Ser Asp Ile Gln Met Thr Gln Ser Pr - #o Ser Ser Leu Ser Ala           #           10                                                                - AGC GTA GGT GAC AGA GTG ACC ATC ACC TGT CG - #A GCA AGC GAG ATT ATT          144                                                                          Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ar - #g Ala Ser Glu Ile Ile           #     25                                                                      - TAC AGT TAT TTA GCA TGG TAC CAG CAG AAG CC - #A GGA AAG GCT CCA AAG          192                                                                          Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pr - #o Gly Lys Ala Pro Lys           # 45                                                                          - CTG CTG ATC TAC AAT GCA AAA ACC TTA GCA GA - #T GGA GTG CCA AGC AGA          240                                                                          Leu Leu Ile Tyr Asn Ala Lys Thr Leu Ala As - #p Gly Val Pro Ser Arg           #                 60                                                          - TTC AGC GGT AGC GGT AGC GGT ACC GAC TAC AC - #C TTC ACC ATC AGC AGC          288                                                                          Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Th - #r Phe Thr Ile Ser Ser           #             75                                                              - CTC CAG CCA GAG GAC ATC GCT ACC TAC TAC TG - #C CAA CAT CAT TTT GGT          336                                                                          Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cy - #s Gln His His Phe Gly           #         90                                                                  - TTT CCT CGG ACG TTC GGC CAA GGG ACC AAG GT - #C GAA ATC AAA                 # 378                                                                         Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Va - #l Glu Ile Lys                   #    105                                                                      #              379                                                            - (2) INFORMATION FOR SEQ ID NO:77:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 126 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                                - Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Va - #l Ala Thr Ala Thr Gly         5                                                                             - Val His Ser Asp Ile Gln Met Thr Gln Ser Pr - #o Ser Ser Leu Ser Ala         #           10                                                                - Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ar - #g Ala Ser Glu Ile Ile         #     25                                                                      - Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pr - #o Gly Lys Ala Pro Lys         # 45                                                                          - Leu Leu Ile Tyr Asn Ala Lys Thr Leu Ala As - #p Gly Val Pro Ser Arg         #                 60                                                          - Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Th - #r Phe Thr Ile Ser Ser         #             75                                                              - Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cy - #s Gln His His Phe Gly         #         90                                                                  - Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Va - #l Glu Ile Lys                 #    105                                                                      __________________________________________________________________________

We claim:
 1. A method to treat an inflammatory condition mediated byinterleukin-8 (IL-8) in a mammalian subject, comprising the step ofadministering to a subject in need of such treatment an amount of areshaped antibody or a fragment of said antibody that specifically bindshuman IL-8 effective to neutralize IL-8 and to ameliorate or modulatesaid inflammatory condition, wherein said antibody comprises two lightchain variable regions comprising the amino acid sequence of SEQ ID NO:73 (RVLa) or SEQ ID NO: 77 (RVLb) and two heavy chain variable regionscomprising the amino acid sequence of SEQ ID NO: 63 (RVHg).
 2. Themethod according to claim 1, wherein said antibody further compriseshuman light chain constant regions and human heavy chain constantregions.
 3. The method according to claim 1, wherein said fragment isF_(ab), F_(ab'), F.sub.(ab')2, or single chain F_(v).